Methods for treating cancer by inhibiting Wnt signaling

ABSTRACT

This invention relates to methods of inhibiting the growth of cancer cells that overexpress a Wnt protein. The methods comprise contacting the cell with an agent that inhibits binding of the Wnt protein to a Frizzled receptor.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional applicationNo. 60/491,350 filed Jul. 31, 2003 and claims benefit of U.S.provisional application No. ______ filed Oct. 4, 2002 (converted fromnon-provisional application Ser. No. 10/264,825). Each application isincorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to methods of inhibiting the growth ofcancer cells that overexpress a Wnt protein. The methods comprisecontacting the cell with an agent that inhibits binding of the Wntprotein to a Frizzled receptor.

BACKGROUND OF THE INVENTION

[0003] The Wingless-type (Wnt) Frizzled protein receptor pathwayinvolves important regulatory genes that carry polymorphisms associatedwith primary carcinomas. In the course of downstream signaling cytosolicβ-catenin accumulates, translocates into the nucleus, and then enhancesgene expression by complexing with other transcription factors Uthoff etal., Mol Carcinog, 31:56-62 (2001). In the absence of Wnt signals, freecytosolic β-catenin is incorporated into a complex consisting of Axin,the adenomatous polyposis coli (APC) gene product, and glycogen synthasekinase (GSK)-3β. Conjunctional phosphorylation of Axin, APC, andβ-catenin by GSK-3β designates β-catenin for the ubiquitin pathway anddegradation by proteasomes Uthoff et al., Mol Carcinog, 31:56-62 (2001);Matsuzawa et al., Mol Cell, 7:915-926 (2001).

[0004] Disheveled (Dvl) is a positive mediator of Wnt signallingpositioned downstream of the frizzled receptors and upstream ofβcatenin. GSK-3 phosphorylates several proteins in the Wnt pathway andis instrumental in the downstream regulation of βcatenin. Mutations inthe gene APC are an initiating event for both sporadic and hereditarycolorectal tumorigenesis. APC mutants are relevant in tumorigenesis,since the aberrant protein is an integral part of the Wnt-signalingcascade. The protein product contains several functional domains actingas binding and degradation sites for βcatenin. Mutations that occur inthe amino-terminal segment of βcatenin are usually involved inphosphorylation-dependent, ubiquitin-mediated degradation and, thus,stabilize βcatenin. When stabilized cytoplasmic-catenin accumulates, ittranslocates to the nucleus interacting with the Tcf/Lef high-mobilitygroup of transcription factors that modulate expression of oncogenessuch as c-myc.

[0005] It is known that Wnt/β-catenin signaling promotes cell survivalin various cell types Orford et al., J Cell Biol, 146:855-868 (1999);Cox et al., Genetics, 155:1725-1740 (2000); Reya et al., Immunity,13:15-24 (2000); Satoh et al., Nat Genet, 24:245-250 (2000); Shin etal., Journal of Biological Chemistry, 274:2780-2785 (1999); Chen et al.,J Cell Biol, 152:87-96 (2001); Ioannidis et al., Nat Immunol, 2:691-697(2001). Wnt signaling pathway is also thought to be associated withtumor development and/or progression (Polakis et al., Genes Dev,14:1837-1851 (2000); Cox et al., Genetics, 155:1725-1740 (2000); Bienzet al., Cell, 103:311-320 (2000); You et al., J Cell Biol, 157:429-440(2002)). Aberrant activation of the Wnt signaling pathway is associatedwith a variety of human cancers, correlating with the over-expression oramplification of c-Myc (Polakis et al., Genes Dev, 14:1837-1851 (2000);Bienz et al., Cell, 103:311-320 (2000); Brown et al., Breast Cancer Res,3:351-355 (2001); He et al., Science, 281:1509-1512 (1998); Miller etal., Oncogene, 18:7860-7872 (1999). In addition, c-Myc was identified asone of the transcriptional targets of the β-catenin/Tcf in colorectalcancer cells (He et al., Science, 281:1509-1512 (1998); de La Coste etal., Proc Natl Acad Sci USA, 95:8847-8851 (1998); Miller et al.,Oncogene, 18:7860-7872 (1999); You et al., J Cell Biol, 157:429-440(2002)).

[0006] In addition to the Wnt ligands, a family of secretedFrizzled-related proteins (sFRPs) has been isolated. sFRPs appear tofunction as soluble endogenous modulators of Wnt signaling by competingwith the membrane-spanning Frizzled receptors for the binding ofsecreted Wnt ligands (Melkonyan et al., Proc Natl Acad Sci USA,94:13636-13641 (1997)). sFRPs can either antagonize Wnt function bybinding the protein and blocking access to its cell surface signalingreceptor, or they can enhance Wnt activity by facilitating thepresentation of ligand to the Frizzled receptors Uthoff et al., MolCarcinog, 31:56-62 (2001). Another protein called Dickkopf (Dkk) is alsofound to interfere with Wnt signaling and diminish accumulation ofcytosolic β-catenin (Fedi et al., J Biol Chem, 274:19465-19472 (1999);Moon et al., Cell, 88:725-728 (1997)). Dkk-1 antagonizes Wnt-inducedsignals by binding to a LDL-receptor-related protein 6 (LRP6) adjacentto the Frizzled receptor (Nusse et al., Nature, 411:255-256 (2001)).Recently H. Suzuki, et al. found that sFRPs are hypermethylated with ahigh frequency in colorectal cancer cell lines and this hypermethylationis associated with a lack of basal sFRP expression (Suzuki et al., NatGenet, 31:141-149 (2002)). Over-expression of Dkk-1 is also found tosensitize brain tumor cells to apoptosis (Shou et al., Oncogene,21:878-889 (2002)).

[0007] Despite recent advances in the understanding of Wnt signaling,the role of this pathway in oncogenesis is unclear. Thus, the prior artfails to provide clear evidence that compounds that modulate thispathway could be useful for treatment of cancer. The present inventionaddresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

[0008] This invention provides methods of inhibiting the growth of acancer cell that overexpresses a Wnt protein. The methods comprisingcontacting the cell with an agent that inhibits binding of the Wntprotein to a Frizzled receptor.

[0009] In some embodiments, the agent is an antibody. For example, theantibody can specifically binds to a Wnt protein, Wnt-1 or Wnt-2. Inother embodiments the antibody specifically binds a Frizzled receptor,such as Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5,Frizzled6, Frizzled7, Frizzled8, Frizzled9 and Frizzled10 receptor.

[0010] Antibodies of the invention can be monoclonal antibodies and canbe prepared and modified in a number of ways. For example, the antibodymay be recombinantly produced. In some embodiments, the antibody is ahumanized antibody or a single chain Fv fragment (scFv).

[0011] The invention also provides therapeutic methods of treatingcancer. In these embodiments, the cancer cell is in a patient and thestep of contacting is carried out by administering the agent to thepatient. The method may further comprise administering to the patient asecond therapeutic agent, such as a chemotherapeutic agent or radiationtherapy. The cancer cell may be a breast cancer cell, colorectal cancercell, a lung cancer cell, a sarcoma cell, or a mesothelioma cell, aprostate cancer cell, a pancreatic cancer cell, a cervical cancer cell,an ovary cancer cell, a gastric cancer cell, an esophageal cancer cell,a head and neck cancer cell, a hepatocellular carcinoma cell, a melanomacell, a glioma cell, or a glioblastoma cell.

[0012] The invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and a monoclonalantibody that specifically binds Wnt a Wnt or Frizzled protein, forexample a Wnt1 protein. The antibody can be further conjugated to aneffector component, such as label, a radioisotope or a cytotoxicchemical.

[0013] In another aspect, the invention provides a method of screeningfor an agent that inhibits the proliferation of a cancer cell, themethod comprising contacting the agent with a Dvl protein or nucleicacid, determining Dvl protein activity or expression, and identifying acompound that inhibits Dvl protein acitivty or expression, therebyidentifying an agent that inhibits the proliferation of a cancer cell.The method can further comprise contacting an identified compound with acancer cell, and selecting the compound that inhibits proliferation ofthe cancer cell. In some embodiments, the cancer cell is a lung cancercell or a mesothelioma cell.

[0014] The invention also provides a method of inhibiting the growth ofa cancer cell that overexpresses a Dvl protein, the method comprisingcontacting the cell with an agent that inhibits Dvl expression oractivity. In some embodiments, the cancer cell is a lung cancer cell ora mesothelioma cell. The agent can be, e.g., a small molecule or ansiRNA.

[0015] Definitions

[0016] The terms “Wnt protein” or “Wnt ligand” refer to a family ofmammalian proteins related to the Drosophila segment polarity gene,wingless. In humans, the Wnt family of genes typically encode 38 to 43kDa cysteine rich glycoproteins having hydrophobic signal sequence, anda conserved asparagine-linked oligosaccharide consensus sequence (seee.g., Shimizu et al Cell Growth Differ 8:1349-1358 (1997)). The Wntfamily contains at least 16 mammalian members. Exemplary Wnt proteinsinclude Wnt-1, Wnt-2, Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-6, Wnt-7A,Wnt-7B, Wnt-8A, Wnt-8B, Wnt-10B, Wnt-11, Wnt-13, Wnt 14, Wnt 15, and Wnt16. The sequence of some exemplary Wnt proteins of the invention are setforth in the sequence listing. In addition, overexpression of particularWnt proteins have been shown to be associated with certain cancers. Forexample, WNT-2 is overexpressed in gastric and colorectal cancer, (Katohet al., Int J Oncol, 19:1003-1007 (2001)); Wnt-1 is overexpressed inhead and neck cancer, and WNT-5A and Wnt-8B are overexpressed in gastriccancer, (Saitoh et al., Int J Mol Med, 9:515-519 (2002); Saitoh et al.,Int J Oncol, 20:343-348 (2002)).

[0017] The terms “frizzled protein” or “frizzled receptor” refer to afamily of mammalian proteins related to the Drosophila frizzled genes,which play a role in the development of tissue polarity. The Frizzledfamily comprises at least 10 mammalian genes. Exemplary human Frizzledreceptors include Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5,Frizzled6, Frizzled7, Frizzled8, Frizzled9 and Frizzled10. The sequenceof exemplary Frizzled receptors are set forth in the sequence listing.The mammalian homologues of the Drosophila frizzled protein share anumber of common structural motifs. The N terminus located at theextracellular membrane surface is followed by a signal sequence, adomain of 120 amino acids with an invariant pattern of 10 cysteineresidues, and a highly divergent region of 40-100 largely variablehydrophilic amino acids. Putative hydrophobic segments form sevenmembrane-spanning helices linked by hydrophilic loops, ending with the Cterminus located at the intracellular face of the membrane. Thecysteine-rich domains (CRDs) and the transmembrane segments are stronglyconserved, suggesting a working model in which an extracellular CRD istethered by a variable linker region to a bundle of sevenmembrane-spanning-helices. Frizzled protein receptors are, therefore,involved in a dynamic model of transmembrane signal transductionanalogous to G-protein-coupled receptors with amino-terminal ligandbinding domains. Frizzled1, Frizzled2, and Frizzled7 in lung andcolorectal cancers, Sagara et al., Commun, 252:117-122 (1998); Frizzled3in human cancer cells including lung, cervical and colorectal cancers,(Kirikoshi et al., Biochem Biophys Res Commun, 271:8-14 (2000));Frizzled7 in gastric cancer (Kirikoshi et al., Int J Oncol, 19:111-115(2001)); Frizzled10 in gastric and colorectal cancer (Kirikoshi et al.,Int J Oncol, 19:767-771 (2001); Terasaki et al., Int J Mol Med,9:107-112 (2002)).

[0018] In addition to the Wnt ligands, a family of secretedfrizzled-related proteins (sFRPs) has been isolated. sFRPs appear tofunction as soluble endogenous modulators of Wnt signaling by competingwith the membrane-spanning frizzled receptors for the binding ofsecreted Wnt ligands. sFRPs, therefore, modulate apoptosissusceptibility, exerting an antagonistic effect on programmed celldeath. sFRPs can either antagonize Wnt function by binding the proteinand blocking access to its cell surface signaling receptor, or they canenhance Wnt activity by facilitating the presentation of ligand to thefrizzled receptors. To date, sFRPs have not yet been linked causativelyto cancer.

[0019] The tern “Dishevelled” or “Dvl” refer to a member of a family ofDishevelled proteins, the full-length sequences of which typicallypossess three conserved domains, a DIX domain, present in the Wntantagonizing protein Axin; a PDZ domain involved in protein-proteininteractions, and a DEP domain found in proteins that regulate RhoGTPases. Dvl proteins include, for example, Dvl-1, Dvl-2, and Dvl-3.Nucleic acid and protein Dvl sequence are known from a variety ofspecies, including mouse and human. Exemplary human Dvl-1, Dvl-2, andDvl-3 protein sequences are available under reference sequencesNP_(—)004412, NP_(—)004413, and NM_(—)004414, respectively.

[0020] “Inhibitors” of Wnt signaling refers to compounds that, e.g.,bind to Wnt or Frizzled proteins, or partially or totally block Wntsignaling as measured in known assays for Wnt signaling (e.g.,measurement of β catenin levels, or oncogene expression controlled byTcf and Lef transcription factors). Inhibitors, include modifiedversions of Wnt or Frizzled proteins, as well as naturally occurring andsynthetic ligands, antagonists, agonists, antibodies, small chemicalmolecules, and the like. Assays for detecting inhibitors of theinvention are described in more detail below.

[0021] A “cancer cell that overexpresses a Wnt protein” is a cancer cellin which expression of a particular Wnt protein is at least about 2times, usually at least about 5 times the level of expression in anormal cell from the same tissue. Methods for determining the level ofexpression of a particular gene are well known in the art. Such methodsinclude RT-PCR, use of antibodies against the gene products, and thelike.

[0022] As used herein, “antibody” includes reference to animmunoglobulin molecule immunologically reactive with a particularantigen, and includes both polyclonal and monoclonal antibodies. Theterm also includes genetically engineered forms such as chimericantibodies (e.g., humanized murine antibodies) and heteroconjugateantibodies (e.g., bispecific antibodies). The term “antibody” alsoincludes antigen binding forms of antibodies, including fragments withantigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG. Seealso, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.). See also, e.g., Kuby, J., Immunology, 3^(rd) Ed., W. H.Freeman & Co., New York (1998). The term also refers to recombinantsingle chain Fv fragments (scFv). The term antibody also includesbivalent or bispecific molecules, diabodies, triabodies, andtetrabodies. Bivalent and bispecific molecules are described in, e.g.,Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992)Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.(1994) J Immunol:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al.(1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, andMcCartney, et al. (1995) Protein Eng. 8:301.

[0023] An antibody immunologically reactive with a particular antigencan be generated by recombinant methods such as selection of librariesof recombinant antibodies in phage or similar vectors, see, e.g., Huseet al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

[0024] Typically, an immunoglobulin has a heavy and light chain. Eachheavy and light chain contains a constant region and a variable region,(the regions are also known as “domains”). Light and heavy chainvariable regions contain four “framework” regions interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework regions and CDRs have beendefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

[0025] The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

[0026] References to “V_(H)” or a “VH” refer to the variable region ofan immunoglobulin heavy chain of an antibody, including the heavy chainof an Fv, scFv, or Fab. References to “V_(L)” or a “VL” refer to thevariable region of an immunoglobulin light chain, including the lightchain of an Fv, scFv, dsFv or Fab.

[0027] The phrase “single chain Fv” or “scFv” refers to an antibody inwhich the variable domains of the heavy chain and of the light chain ofa traditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site.

[0028] A “chimeric antibody” is an immunoglobulin molecule in which (a)the constant region, or a portion thereof, is altered, replaced orexchanged so that the antigen binding site (variable region) is linkedto a constant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0029] A “humanized antibody” is an immunoglobulin molecule whichcontains minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, a humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

[0030] “Epitope” or “antigenic determinant” refers to a site on anantigen to which an antibody binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0031] “Biological sample” as used herein is a sample of biologicaltissue or fluid that contains nucleic acids or polypeptides, e.g., of aWnt protein, polynucleotide or transcript. Such samples include, but arenot limited to, tissue isolated from primates, e.g., humans, or rodents,e.g., mice, and rats. Biological samples may also include sections oftissues such as biopsy and autopsy samples, frozen sections taken forhistologic purposes, blood, plasma, serum, sputum, stool, tears, mucus,hair, skin, etc. Biological samples also include explants and primaryand/or transformed cell cultures derived from patient tissues. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

[0032] “Providing a biological sample” means to obtain a biologicalsample for use in methods described in this invention. Most often, thiswill be done by removing a sample of cells from an animal, but can alsobe accomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues,having treatment or outcome history, will be particularly useful.

[0033] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions, aswell as naturally occurring, e.g., polymorphic or allelic variants, andman-made variants. As described below, the preferred algorithms canaccount for gaps and the like. Preferably, identity exists over a regionthat is at least about 25 amino acids or nucleotides in length, or morepreferably over a region that is 50-100 amino acids or nucleotides inlength.

[0034] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0035] A “comparison window”, as used herein, includes reference to asegment of one of the number of contiguous positions selected from thegroup consisting typically of from 20 to 600, usually about 50 to about200, more usually about 100 to about 150 in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequences for comparison are well-known in the art. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds. 1995 supplement)).

[0036] Preferred examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity includethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol.Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, e.g.,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0037] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

[0038] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, e.g., where the two peptides differonly by conservative substitutions. Another indication that two nucleicacid sequences are substantially identical is that the two molecules ortheir complements hybridize to each other under stringent conditions, asdescribed below. Yet another indication that two nucleic acid sequencesare substantially identical is that the same primers can be used toamplify the sequences.

[0039] The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure. “Purify” or“purification” in other embodiments means removing at least onecontaminant from the composition to be purified. In this sense,purification does not require that the purified compound be homogenous,e.g., 100% pure.

[0040] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

[0041] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

[0042] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0043] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

[0044] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention. Typicallyconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0045] Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3rd ed., 1994) and Cantor & Schimmel, BiophysicalChemistry Part I: The Conformation of Biological Macromolecules (1980).“Primary structure” refers to the amino acid sequence of a particularpeptide. “Secondary structure” refers to locally ordered, threedimensional structures within a polypeptide. These structures arecommonly known as domains. Domains are portions of a polypeptide thatoften form a compact unit of the polypeptide and are typically 25 toapproximately 500 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of (-sheet and(-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed, usually by the noncovalentassociation of independent tertiary units. Anisotropic terms are alsoknown as energy terms.

[0046] A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include fluorescentdyes, electron-dense reagents, enzymes (e.g., as commonly used in anELISA), biotin, digoxigenin, or haptens and proteins or other entitieswhich can be made detectable, e.g., by incorporating a radiolabel intothe peptide or used to detect antibodies specifically reactive with thepeptide. The radioisotope may be, for example, 3H, 14C, 32P, 35S, or125I. In some cases, particularly using antibodies against the proteinsof the invention, the radioisotopes are used as toxic moieties, asdescribed below. The labels may be incorporated into the nucleic acids,proteins and antibodies at any position. Any method known in the art forconjugating the antibody to the label may be employed, including thosemethods described by Hunter et al., Nature, 144:945 (1962); David etal., Biochemistry, 13:1014 (1974); Pain et al., J Immunol. Meth., 40:219(1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982). Thelifetime of radiolabeled peptides or radiolabeled antibody compositionsmay extended by the addition of substances that stablize theradiolabeled peptide or antibody and protect it from degradation. Anysubstance or combination of substances that stablize the radiolabeledpeptide or antibody may be used including those substances disclosed inU.S. Pat. No. 5,961,955.

[0047] An “effector” or “effector moiety” or “effector component” is amolecule that is bound (or linked, or conjugated), either covalently,through a linker or a chemical bond, or noncovalently, through ionic,van der Waals, electrostatic, or hydrogen bonds, to an antibody. The“effector” can be a variety of molecules including, e.g., detectionmoieties including radioactive compounds, fluorescent compounds, anenzyme or substrate, tags such as epitope tags, a toxin; activatablemoieties, a chemotherapeutic agent; a lipase; an antibiotic; or aradioisotope emitting “hard” e.g., beta radiation.

[0048] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not. normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

[0049] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

[0050] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein, in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein sequences at least two times the background and more typicallymore than 10 to 100 times background.

[0051] Specific binding to an antibody under such conditions requires anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to a particular protein,polymorphic variants, alleles, orthologs, and conservatively modifiedvariants, or splice variants, or portions thereof, can be selected toobtain only those polyclonal antibodies that are specificallyimmunoreactive with Wnt or Frizzled proteins and not with otherproteins. This selection may be achieved by subtracting out antibodiesthat cross-react with other molecules. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

[0052] “Tumor cell” refers to precancerous, cancerous, and normal cellsin a tumor.

[0053] “Cancer cells,” “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. In the present invention transformation istypically associated with overexpression of Wnt and/or Frizzledproteins. Transformation is associated with other phenotypic changes,such as immortalization of cells, aberrant growth control,nonmorphological changes, and/or malignancy (see, Freshney, Culture ofAnimal Cells a Manual of Basic Technique (3rd ed. 1994)).

BRIEF DESCRIPTION OF THE DRAWINGS

[0054]FIG. 1 shows that anti-Wnt-1 or anti-Wnt-2 antibody specificallyinduces apoptosis in various human cancer cell lines.

[0055]FIG. 2 shows the fraction of apoptotic cell death (%) afteranti-Wnt antibody treatment.

[0056]FIG. 3A shows that anti-Wnt antibody-induced apoptosis iscorrelated with the Wnt expression in various cancer cell lines. FIG. 3Bshows the effect of Wnt blocking peptides on anti-Wnt antibody-inducedapoptosis.

[0057]FIG. 4 shows a time course (FIG. 4A) and dosage cures of anti-Wntantibody-induced apoptosis in lung cancer cell lines (FIG. 4B).

[0058]FIG. 5 shows that anti-Wnt-1 monoclonal antibody induces apoptosisin different human cancer cell lines in vitro. a. 0.5% Crystal Violetstaining of cancer cells MCF-7 (upper two rows) about 48 hrs and H460(bottom two rows) about 72 hrs after control or the anti-Wnt-1monoclonal antibody treatment. Concentrations of the control oranti-Wnt-1 antibodies used from left to right are 0.0, 1.0 and 10.0μg/ml, respectively. b. Example of apoptosis analysis by flow cytometry.From top to bottom, H460 cancer cells were treated with 5.0 μg/ml ofcontrol antibody, 1.0 μg/ml and 5.0 μg/ml of anti-Wnt-1 antibody,respectively, for about 72 hrs. FL1-H represents Annexin V-FITC stainingand FL3-H represents propidium iodide (PI) staining. c. Dose responsesof H460 and MCF-7 cancer cells to monoclonal antibody treatment.Measurements were taken after 72 hrs of incubation for H460 and 48 hrsof incubation for MCF-7. Squares (□) and circles (∘) represent fractionof cell death in MCF-7 and H460 cells treated with anti-Wnt-1 antibody,respectively. Diamonds (⋄) and triangles (Δ) represent fraction of celldeath in MCF-7 and H460 cells treated with control antibody,respectively. Results are the means±SD (error bars).

[0059]FIGS. 6A-6C show that an anti-Wnt-1 monoclonal antibody suppressestumor growth in vivo.

[0060]FIG. 7 shows the sequences of heavy and light chain regions ofmonoclonal antibodies generated to peptides equences set forth in SEQ IDNO:2, SEQ ID NO:4, or SEQ ID NO:9.

[0061]FIG. 8 shows that ΔPDZ-Dvl inhibited the tumorigenesis ofmesothelioma cell in vivo. ΔPDZ-Dvl-transfected malignant pleuralmesothelioma LRK1A and REN cells were unable to grow after subcutaneous(s.c.) injection in athymic mice compared with empty vector-trasnfectedcontrols. Results are the means±SD (bars) for five animals in eachgroup.

[0062]FIG. 9 shows suppression of NCI-H1703 growth by the Dvl siRNA.Cells (3×10⁴) were plated in 24-2311 plates and transfected with the DvlsiRNA (sequares) or the contro si RNA (circles). After 72 h oftransfection, viable cells (trypan blue exclusion were collected every24 h by trypsinization and counted. Notably, after 72 h of transfection,cell growth was significantly suppressed (P<0.05).

[0063]FIG. 10 shows that over-expression of Wnt signal antagonist FRP orDKK induces apoptosis in cancer cells.

DETAILED DESCRIPTION

[0064] This invention is based on the discovery that Wnt-Fz signalingpathway plays a role in oncogenesis. It is known that Wnt proteins oftenhave high level expression in cancer. However, little is known regardingWnt-Fz signaling modification of the cell death machinery in cancer. Thepresent disclosure provides evidence that inhibitors of Wnt signalingcan induce significant apoptosis in a number of cancer cells. Theinvention is useful for any cancer in which Wnt-Fz signaling affectscancer cell growth or survival. The invention is useful for treatingcancers such as breast cancer, colorectal cancer, lung cancer, sarcoma,mesothelioma, prostate cancer, pancreatic cancer,cervical cancer,ovarian cancer, gastric cancer, esophageal cancer, head and neck cancer,hepatocellular carcinoma, melanoma, glioma, or glioblastoma.

[0065] Blocking Wnt signaling is shown here to lead to down-regulationof downstream components of the Wnt-Fz pathway, in particular,Dishevelled (Dvl) and β-catenin. Evidence provided here also shows thatantibody-induced apoptosis occurs through activation of JNK, releasingSmac/Diablo and cytochrome C from mitochondria to the cytosol.Cytochrome C inactivates survinin, an inhibitor of apoptosis, that leadsto the activation of caspases. The disclosure further provides evidencethat monoclonal anti-Wnt-1 antibodies can suppress growth of tumors invivo.

[0066] Antibodies to Wnt and Frizzled Proteins

[0067] As noted above, the invention provides methods of inhibiting Wntsignaling in cancer cells. In some embodiments of the invention,antibodies are used to block the binding between Wnt ligand and theFrizzled receptor. The antibodies can be raised against either Wnt orFrizzled proteins.

[0068] Methods of preparing polyclonal antibodies are known to theskilled artisan (e.g., Coligan, supra; and Harlow & Lane, supra).Polyclonal antibodies can be raised in a mammal, e.g., by one or moreinjections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include a protein encoded by a nucleic acid of thefigures or fragment thereof or a fusion protein thereof It may be usefulto conjugate the immunizing agent to a protein known to be immunogenicin the mammal being immunized. Examples of such immunogenic proteinsinclude but are not limited to keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, and soybean trypsin inhibitor. Examples ofadjuvants which may be employed include Freund's complete adjuvant andMPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

[0069] The antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler & Milstein, Nature 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro. The immunizing agent will typically include apolypeptide encoded by a nucleic acid of Tables 1-16 fragment thereof,or a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (1986)). Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

[0070] In some embodiments, a monoclonal antibody is used. A preferredembodiment is a monoclonal antibody that binds the same epitope as themonoclonal antibody described in Example 11. The ability of a particularantibody to recognize the same epitope as another antibody is typicallydetermined by the ability of one antibody to competitively inhibitbinding of the second antibody to the antigen. Any of a number ofcompetitive binding assays can be used to measure competition betweentwo antibodies to the same antigen. For example, a sandwich ELISA assaycan be used for this purpose. This is carried out by using a captureantibody to coat the surface of a well. A subsaturating concentration oftagged-antigen is then added to the capture surface. This protein willbe bound to the antibody through a specific antibody:epitopeinteraction. After washing a second antibody, which has been covalentlylinked to a detectable moeity (e.g., HRP, with the labeled antibodybeing defined as the detection antibody) is added to the ELISA. If thisantibody recognizes the same epitope as the capture antibody it will beunable to bind to the target protein as that particular epitope will nolonger be available for binding. If however this second antibodyrecognizes a different epitope on the target protein it will be able tobind and this binding can be detected by quantifying the level ofactivity (and hence antibody bound) using a relevant substrate. Thebackground is defined by using a single antibody as both capture anddetection antibody, whereas the maximal signal can be established bycapturing with an antigen specific antibody and detecting with anantibody to the tag on the antigen. By using the background and maximalsignals as references, antibodies can be assessed in a pair-wise mannerto determine epitope specificity.

[0071] A first antibody is considered to competitively inhibit bindingof a second antibody, if binding of the second antibody to the antigenis reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays described above.

[0072] In some embodiments, a monoclonal anti-Wnt antibody of theinvention binds to amino acids 201-212 of human Wnt-1 (HNNEAGRTTVFS),amino acids 39-52 of human Wnt-1 (NVASSTNLLTDSKS), or amino acids 49-63of human Wnt-2 (SSQRQLCHRHPDVMR). For example, such a monoclonalantibody may have the binding specificity (i.e., in this context, thesame CDRs, or substantially the same CDRs) of an antibody having V_(H)and V_(L) chains as set forth in FIG. 7. An antibody of the inventionmay therefore comprises a CDR as set forth in a V_(H) or V_(L) sequenceshown in FIG. 7 and, additionally, may have at least 80% identity,preferably, 85%, 90%, or 95% identity to the V_(H) or V_(L) sequence.For example, in particular embodiments, the antibody may comprise theCDRs of a V_(H) and V_(L) sequence of FIG. 7 and human frameworksequences.

[0073] In some embodiments the antibodies to the Wnt or Frizzledproteins are chimeric or humanized antibodies. As noted above, humanizedforms of antibodies are chimeric immunoglobulins in which residues froma complementary determining region (CDR) of human antibody are replacedby residues from a CDR of a non-human species such as mouse, rat orrabbit having the desired specificity, affinity and capacity.

[0074] Human antibodies can be produced using various techniques knownin the art, including phage display libraries (Hoogenboom & Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, p. 77 (1985) and Boemer et al., J.Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be madeby introducing of human immunoglobulin loci into transgenic animals,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg& Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0075] In some embodiments, the antibody is a single chain Fv (scFv).The V_(H) and the V_(L) regions of a scFv antibody comprise a singlechain which is folded to create an antigen binding site similar to thatfound in two chain antibodies. Once folded, noncovalent interactionsstabilize the single chain antibody. While the V_(H) and V_(L) regionsof some antibody embodiments can be directly joined together, one ofskill will appreciate that the regions may be separated by a peptidelinker consisting of one or more amino acids. Peptide linkers and theiruse are well-known in the art. See, e.g., Huston et al., Proc. Nat'lAcad. Sci. USA 8:5879 (1988); Bird et al., Science 242:4236 (1988);Glockshuber et al., Biochemistry 29:1362 (1990); U.S. Pat. No.4,946,778, U.S. Pat. No. 5,132,405 and Stemmer et al., Biotechniques14:256-265 (1993). Generally the peptide linker will have no specificbiological activity other than to join the regions or to preserve someminimum distance or other spatial relationship between the V_(H) andV_(L). However, the constituent amino acids of the peptide linker may beselected to influence some property of the molecule such as the folding,net charge, or hydrophobicity. Single chain Fv (scFv) antibodiesoptionally include a peptide linker of no more than 50 amino acids,generally no more than 40 amino acids, preferably no more than 30 aminoacids, and more preferably no more than 20 amino acids in length. Insome embodiments, the peptide linker is a concatamer of the sequenceGly-Gly-Gly-Gly-Ser, preferably 2, 3, 4, 5, or 6 such sequences.However, it is to be appreciated that some amino acid substitutionswithin the linker can be made. For example, a valine can be substitutedfor a glycine.

[0076] Methods of making scFv antibodies have been described. See, Huseet al., supra; Ward et al. supra; and Vaughan et al., supra. In brief,mRNA from B-cells from an immunized animal is isolated and cDNA isprepared. The cDNA is amplified using primers specific for the variableregions of heavy and light chains of immunoglobulins. The PCR productsare purified and the nucleic acid sequences are joined. If a linkerpeptide is desired, nucleic acid sequences that encode the peptide areinserted between the heavy and light chain nucleic acid sequences. Thenucleic acid which encodes the scFv is inserted into a vector andexpressed in the appropriate host cell. The scFv that specifically bindto the desired antigen are typically found by panning of a phage displaylibrary. Panning can be performed by any of several methods. Panning canconveniently be performed using cells expressing the desired antigen ontheir surface or using a solid surface coated with the desired antigen.Conveniently, the surface can be a magnetic bead. The unbound phage arewashed off the solid surface and the bound phage are eluted.

[0077] Regardless of the method of panning chosen, the physical linkbetween genotype and phenotype provided by phage display makes itpossible to test every member of a cDNA library for binding to antigen,even with large libraries of clones.

[0078] In some embodiments, the antibodies are bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens or that have binding specificities for two epitopes on the sameantigen. In one embodiment, one of the binding specificities is for theWnt or Frizzled protein, the other one is for another cancer antigen.Alternatively, tetramer-type technology may create multivalent reagents.

[0079] In some embodiments, the antibody is conjugated to an effectormoiety. The effector moiety can be any number of molecules, includinglabeling moieties such as radioactive labels or fluorescent labels, orcan be a therapeutic moiety. If the effector moiety is a therapeuticmoiety, it will typically be a cytotoxic agent. In this method,targeting the cytotoxic agent to cancer cells, results in direct killingof the target cell. This embodiment is typically carried out usingantibodies against the Frizzled receptor. Cytotoxic agents are numerousand varied and include, but are not limited to, cytotoxic drugs ortoxins or active fragments of such toxins. Suitable toxins and theircorresponding fragments include diphtheria A chain, exotoxin A chain,ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin,auristatin and the like. Cytotoxic agents also include radiochemicalsmade by conjugating radioisotopes to antibodies raised against Wnt orFrizzled proteins, or binding of a radionuclide to a chelating agentthat has been covalently attached to the antibody.

[0080] Binding Affinity of Antibodies of the Invention

[0081] Binding affinity for a target antigen is typically measured ordetermined by standard antibody-antigen assays, such as Biacorecompetitive assays, saturation assays, or immunoassays such as ELISA orRIA.

[0082] Such assays can be used to determine the dissociation constant ofthe antibody. The phrase “dissociation constant” refers to the affinityof an antibody for an antigen. Specificity of binding between anantibody and an antigen exists if the dissociation constant (K_(D)=1/K,where K is the affinity constant) of the antibody is<1 μM,preferably<100 nM, and most preferably<0.1 nM. Antibody molecules willtypically have a K_(D) in the lower ranges. K_(D)=[Ab-Ag]/(Aberle etal., EMBO Journal, 16:3797-3804 (1997)) where (Aberle et al., EMBOJournal, 16:3797-3804 (1997)) is the concentration at equilibrium of theantibody, (Aberle et al., EMBO Journal, 16:3797-3804 (1997)) is theconcentration at equilibrium of the antigen and [Ab-Ag] is theconcentration at equilibrium of the antibody-antigen complex. Typically,the binding interactions between antigen and antibody include reversiblenoncovalent associations such as electrostatic attraction, Van der Waalsforces and hydrogen bonds.

[0083] The antibodies of the invention specifically bind to Wnt orFrizzled proteins. By “specifically bind” herein is meant that theantibodies bind to the protein with a K_(D) of at least about 0.1 mM,more usually at least about 1 μM, preferably at least about 0.1 μM orbetter, and most preferably, 0.01 μM or better.

[0084] Diagnostic Assays

[0085] The present invention also provides diagnostic assays fordetecting Wnt or Frizzled over-expression. As noted aboveover-expression of these genes can be used to identify cancer cells. Inpreferred embodiments, activity of the Wnt or Frizzled gene of interestis determined by a measure of gene transcript (e.g. MRNA), by a measureof the quantity of translated protein, or by a measure of gene productactivity.

[0086] Methods of detecting and/or quantifying the gene transcript (MRNAor cDNA) using nucleic acid hybridization techniques are known to thoseof skill in the art. For example, one method for evaluating thepresence, absence, or quantity of MRNA involves a Northern blottransfer.

[0087] The probes can be full length or less than the full length of thenucleic acid sequence encoding the protein. Shorter probes areempirically tested for specificity. Preferably nucleic acid probes are20 bases or longer in length. Visualization of the hybridized portionsallows the qualitative determination of the presence or absence of MRNA.

[0088] In another preferred embodiment, a transcript (e.g., MRNA) can bemeasured using amplification (e.g. PCR) based methods as described abovefor directly assessing copy number of DNA. In a preferred embodiment,transcript level is assessed by using reverse transcription PCR(RT-PCR).

[0089] The “activity” of a Wnt or Frizzled gene can also be detectedand/or quantified by detecting or quantifying the expressed polypeptide.The polypeptide can be detected and quantified by any of a number ofmeans well known to those of skill in the art. These may includeanalytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like.The isolated proteins can also be sequence according to standardtechniques to identify polymorphisms.

[0090] The antibodies of the invention can also be used to detect Wnt orFrizzled proteins, or cells expressing them, using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,1 10; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology, Vol. 37, Asai,ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology7th Edition, Stites & Terr, eds. (1991).

[0091] Thus, the present invention provides methods of detecting cellsthat over-express Wnt or Frizzled proteins. In one method, a biopsy isperformed on the subject and the collected tissue is tested in vitro.The tissue or cells from the tissue is then contacted, with an anti-Wntor anti-Frizzled antibody of the invention. Any immune complexes whichresult indicate the presence of the target protein in the biopsiedsample. To facilitate such detection, the antibody can be radiolabeledor coupled to an effector molecule which is a detectable label, such asa radiolabel. In another method, the cells can be detected in vivo usingtypical imaging systems. Then, the localization of the label isdetermined by any of the known methods for detecting the label. Aconventional method for visualizing diagnostic imaging can be used. Forexample, paramagnetic isotopes can be used for MRI. Internalization ofthe antibody may be important to extend the life within the organismbeyond that provided by extracellular binding, which will be susceptibleto clearance by the extracellular enzymatic environment coupled withcirculatory clearance.

[0092] Identification of Inhibitors of Wnt Signaling

[0093] Wnt or Frizzled proteins (or cells expressing them) or members ofthe Wnt signaling pathway, e.g., dvl, can also be used in drug screeningassays to identify agents that inhibit Wnt signaling. The presentinvention thus provides novel methods for screening for compositionswhich inhibit cancer.

[0094] Assays for Wnt signaling can be designed to detect and/orquantify any part of the Wnt signaling pathway. For example the abilityof an agent to affect intracellular β-catenin levels or to induceaoptosis in target cells can be measured. Assays suitable for thesepurposes are described below.

[0095] Assays may include those designed to test binding activity toeither the Wnt ligand, the Frizzled receptor, or another member of theWnt signaling cascade, e.g., dvl. These assays are particularly usefulin identifying agents that modulate Wnt activity. Virtually any agentcan be tested in such an assay. Such agents include, but are not limitedto natural or synthetic polypeptides, antibodies, natural or syntheticsmall organic molecules, nucleic acids and the like.

[0096] As noted above, a family of secreted Frizzled-related proteins(sFRPs) function as soluble endogenous modulators of Wnt signaling bycompeting with Frizzled receptors for the binding of secreted Wntligands. Thus, in some format, test agents are based on natural ligands(e.g., Wnts ligands or sFRPs) of the Frizzled receptor.

[0097] Any of the assays for detecting Wnt signaling are amenable tohigh throughput screening. High throughput assays binding assays andreporter gene assays are similarly well known. Thus, for example, U.S.Pat. No. 5,559,410 discloses high throughput screening methods forproteins, U.S. Pat. No. 5,585,639 discloses high throughput screeningmethods for nucleic acid binding (i.e., in arrays), while U.S. Pat. Nos.5,576,220 and 5,541,061 disclose high throughput methods of screeningfor ligand/antibody binding.

[0098] In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols for various high throughput systems.Thus, for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

[0099] Other assays useful in the present invention are those designedto test neoplastic phenotypes of cancer cells. These assays include cellgrowth on soft agar; anchorage dependence; contact inhibition anddensity limitation of growth; cellular proliferation; cell death(apoptosis); cellular transformation; growth factor or serum dependence;tumor specific marker levels; invasiveness into Matrigel; tumor growthand metastasis in vivo; mRNA and protein expression in cells undergoingmetastasis, and other characteristics of cancer cells.

[0100] The ability of test agents to inhibit cell growth can also beassessed by introducing the test into an animal model of disease, andassessing the growth of cancer cells in vivo. For example, human tumorcells can be introduced into an immunocompromised animal such as a “nudemouse”. The test agent (e.g., a small molecule or an antibody) isadministered to the animal and the ability of the tumor cell to formtumors—as assessed by the number and/or size of tumors formed in theanimal—is compared to tumor growth in a control animal without theagent.

[0101] Inhibitors of Gene Expression

[0102] In one aspect of the present invention, inhibitors of the Wntsignaling pathway, e.g., Dvl inhibitors, can comprise nucleic acidmolecules that inhibit expression of the target protein in the pathway.Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered polypeptides, e.g.,dominant negative forms of the protein, in mammalian cells or targettissues, or alternatively, nucleic acids e.g., inhibitors of targetprotein expression, such as siRNAs or anti-sense RNAs. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology Doerfler and Böhm(eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0103] In some embodiments, small interfering RNAs are administered. Inmammalian cells, introduction of long dsRNA (>30 nt) often initiates apotent antiviral response, exemplified by nonspecific inhibition ofprotein synthesis and RNA degradation. The phenomenon of RNAinterference is described and discussed, e.g., in Bass, Nature411:428-29 (2001); Elbahir et al., Nature 411:494-98 (2001); and Fire etal., Nature 391:806-11 (1998), where methods of making interfering RNAalso are discussed. The siRNA inhibitors are less than 100 base pairs,typically 30 bps or shorter, and are made by approaches known 30 in theart. Exemplary siRNAs according to the invention can have up to 29 bps,25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integerthereabout or therebetween.

[0104] Non-Viral Delivery Methods

[0105] Methods of non-viral delivery of nucleic acids encodingengineered polypeptides of the invention include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described ine.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

[0106] The preparation of lipid:nucleic acid complexes, includingtargeted liposomes such as immunolipid complexes, is well known to oneof skill in the art (see, e.g., Crystal, Science 270:404-410 (1995);Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad etal., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183,4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,4,837,028, and 4,946,787).

[0107] Viral Delivery Methods

[0108] The use of RNA or DNA viral based systems for the delivery ofinhibitors of target Wnt pathway proteins, e.g., Dvl, are known in theart. Conventional viral based systems for the delivery of such nucleicacid inhibitors can include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.

[0109] In many gene therapy applications, it is desirable that the genetherapy vector be delivered with a high degree of specificity to aparticular tissue type, e.g., a lung cancer. A viral vector is typicallymodified to have specificity for a given cell type by expressing aligand as a fusion protein with a viral coat protein on the virusesouter surface. The ligand is chosen to have affinity for a receptorknown to be present on the cell type of interest. For example, Han etal., PNAS 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

[0110] Gene therapy vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient.

[0111] Ex vivo cell transfection for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transfected cells into the hostorganism) is well known to those of skill in the art. In someembodiments, cells are isolated from the subject organism, transfectedwith inhibitor nucleic acids and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

[0112] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can also be administered directlyto the organism for transduction of cells in vivo. Alternatively, nakedDNA can be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

[0113] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention, as described below (see, e.g.,Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0114] Kits Use in Diagnostic, Research, and Therapeutic Applications

[0115] As noted above, the invention provides evidence of theoverexpression of particular Wnt or Frizzled proteins in certaincancers. Thus, kits can be used for the detection of the particularnucleic acids or proteins disclosed here. In diagnostic and researchapplications such kits may include any or all of the following: assayreagents, buffers, Wnt-specific or Frizzled-specific nucleic acids orantibodies, hybridization probes and/or primers, and the like. Atherapeutic product may include sterile saline or anotherpharmaceutically acceptable emulsion and suspension base.

[0116] In addition, the kits may include instructional materialscontaining directions (i.e., protocols) for the practice of the methodsof this invention. While the instructional materials typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

[0117] The present invention also provides for kits for screening forinhibitors of Wnt signaling. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseone or more of the following materials: a Wnt or Frizzled polypeptide orpolynucleotide, reaction tubes, and instructions for testing the desiredWnt signaling function (e.g., β catenin levels).

[0118] Therapeutic Methods

[0119] Administration of Inhibitors

[0120] The agents that inhibit Wnt signaling (e.g., antibodies) can beadministered by a variety of methods including, but not limited toparenteral (e.g., intravenous, intramuscular, intradermal,intraperitoneal, and subcutaneous routes), topical, oral, local, ortransdermal administration. These methods can be used for prophylacticand/or therapeutic treatment.

[0121] As noted above, inhibitors of the invention can be used to treatcancers associated with Wnt signaling. The compositions foradministration will commonly comprise a inhibitor dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike. These solutions are sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like. Theconcentration of active agent in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

[0122] Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when-the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

[0123] The pharmaceutical compositions can be administered in a varietyof unit dosage forms depending upon the method of administration. Forexample, unit dosage forms suitable for oral administration include, butare not limited to, powder, tablets, pills, capsules and lozenges. It isrecognized that antibodies when administered orally, should be protectedfrom digestion. This is typically accomplished either by complexing themolecules with a composition to render them resistant to acidic andenzymatic hydrolysis, or by packaging the molecules in an appropriatelyresistant carrier, such as a liposome or a protection barrier. Means ofprotecting agents from digestion are well known in the art.

[0124] The compositions containing inhibitors of the invention (e.g.,antibodies) can be administered for therapeutic or prophylactictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease (e.g., breast cancer) in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the agents of this invention toeffectively treat the patient. An amount of an inhibitor that is capableof preventing or slowing the development of cancer in a patient isreferred to as a “prophylactically effective dose.” The particular doserequired for a prophylactic treatment will depend upon the medicalcondition and history of the patient, the particular cancer beingprevented, as well as other factors such as age, weight, gender,administration route, efficiency, etc. Such prophylactic treatments maybe used, e.g., in a patient who has previously had cancer to prevent arecurrence of the cancer, or in a patient who is suspected of having asignificant likelihood of developing cancer.

[0125] A “patient” for the purposes of the present invention includesboth humans and other animals, particularly mammals. Thus the methodsare applicable to both human therapy and veterinary applications. In thepreferred embodiment the patient is a mammal, preferably a primate, andin the most preferred embodiment the patient is human.

[0126] Other known cancer therapies can be used in combination with themethods of the invention. For example, inhibitors of Wnt signaling mayalso be used to target or sensitize the cell to other cancer therapeuticagents such as 5FU, vinblastine, actinomycin D, cisplatin, methotrexate,and the like. In other embodiments, the methods of the invention can beused with radiation therapy and the like.

[0127] In some instances the antibody belongs to a sub-type thatactivates serum complement when complexed with the transmembrane proteinthereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC).Thus, cancer can be treated by administering to a patient antibodiesdirected against Frizzled proteins on the surface of cancer cells.Antibody-labeling may activate a co-toxin, localize a toxin payload, orotherwise provide means to locally ablate cells. In these embodiments,the antibody is conjugated to an effector moiety. The effector moietycan be any number of molecules, including labeling moieties such asradioactive labels or fluorescent labels, or can be a therapeuticmoiety, such as a cytotoxic agent.

[0128] Use of Wnt or Frizzled Polypeptides as Vaccines

[0129] In addition to administration of inhibitors of wnt signalling,the Wnt or Frizzled proteins or immunogenic fragments of them can beadministered as vaccine compositions to stimulate HTL, CTL, and antibodyresponses against the endogenous proteins. Such vaccine compositions caninclude, e.g., lipidated peptides (see, e.g., Vitiello, et al. (1995) J.Clin. Invest. 95:341-349), peptide compositions encapsulated inpoly(D,L-lactide-co-glycolide, “PLG”) microspheres (see, e.g., Eldridge,et al. (1991) Molec. Immunol. 28:287-294; Alonso, et al. (1994) Vaccine12:299-306; Jones, et al. (1995) Vaccine 13:675-681), peptidecompositions contained in immune stimulating complexes (ISCOMS; see,e.g., Takahashi, et al. (1990) Nature 344:873-875; Hu, et al. (1998)Clin. Exp. Immunol. 113:235-243), multiple antigen peptide systems(MAPs; see, e.g., Tam (1988) Proc. Nat'l Acad. Sci. USA 85:5409-5413;Tam (1996) J. Immunol. Methods 196:17-32); viral delivery vectors(Perkus, et al., p. 379, in Kaufmann (ed. 1996) Concepts in VaccineDevelopment de Gruyter; Chakrabarti, et al. (1986) Nature 320:535-537;Hu, et al. (1986) Nature 320:537-540; Kieny, et al. (1986) AIDSBio/Technology 4:790-795; Top, et al. (1971) J. Infect. Dis.124:148-154; Chanda, et al. (1990) Virology 175:535-547), particles ofviral or synthetic origin (see, e.g., Kofler, et al. (1996) J. Immunol.Methods 192:25-35; Eldridge, et al. (1993) Sem. Hematol. 30:16-24; Falo,et al. (1995) Nature Med. 7:649-653).

[0130] Vaccine compositions often include adjuvants. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis, orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, e.g., Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

[0131] Vaccines can be administered as nucleic acid compositions whereinDNA or RNA encoding the Wnt or Frizzled polypeptides, or a fragmentthereof, is administered to a patient. See, e.g., Wolff et. al. (1990)Science 247:1465-1468; U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566;5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-baseddelivery technologies include “naked DNA”, facilitated (bupivicaine,polymers, peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

[0132] Methods for the use of genes as DNA vaccines are well known, andinclude placing the desired gene or portion thereof under the control ofa regulatable promoter or a tissue-specific promoter for expression inthe patient. The gene used for DNA vaccines can encode full-length Wntor Frizzled protein, or may encode portions of the proteins.

[0133] In a some embodiments, the DNA vaccines include a gene encodingan adjuvant molecule with the DNA vaccine. Such adjuvant moleculesinclude cytokines that increase the immunogenic response to thepolypeptide encoded by the DNA vaccine.

[0134] For therapeutic or prophylactic immunization purposes, thepeptides of the invention can be expressed by viral or bacterialvectors. Examples of expression vectors include attenuated viral hosts,such as vaccinia or fowlpox. This approach involves the use of vacciniavirus, e.g., as a vector to express nucleotide sequences that encode Wntor Frizzled polypeptides or polypeptide fragments. Upon introductioninto a host, the recombinant vaccinia virus expresses the immunogenicpeptide, and thereby elicits an immune response. Vaccinia vectors andmethods useful in immunization protocols are described in, e.g., U.S.Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCGvectors are described in Stover, et al. (1991) Nature 351:456-460. Awide variety of other vectors useful for therapeutic administration orimmunization e.g., adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent. See, e.g., Shata, et al. (2000) Mol. Med.Today 6:66-71; Shedlock, et al. (2000) J. Leukoc. Biol. 68:793-806; andHipp, et al. (2000) In Vivo 14:571-85.

EXAMPLES

[0135] The following examples are offered to illustrate, but not tolimit the claimed invention.

[0136] Materials and Methods

[0137] Cell Lines

[0138] Human non-small-cell lung cancer (NSCLC) cell lines (NCI-H460,NCI-H838 and NCI-A549), normal lung cell line (CCL-75, fibroblast),human breast cancer cell lines (MCF-7 and SKBR-3), human colon cancercell line SW480, and human mesothelioma cancer cell lines H28 wereobtained from American Type Culture Collections (ATCC) (Manassas, Va.).Other human mesothelioma cancer cell line NCI-H290 was obtained from NIH(Frederick, Md.) and REN was kindly provided by Dr. Steven Albelda's labat the University of Pennsylvania (Philadelphia, Pa.). Normalmesothelial cell line LP-9 was obtained from the Cell Culture CoreFacility at Harvard University (Boston, Mass.). Human osteosarcomacancer cell line Saos-2 was obtained from the Cell Culture Facility atUCSF. Mouse mammary cell lines: C57MG transfected with empty-vector(C57MG) and transfected with Wnt-1 (C57Wnt-1) were kindly provided byDr. Frank McCormick's Lab at UCSF Cancer Center. These cells, exceptCCL-75, LP-9, and Saos-2, were cultured in RPMI 1640 supplemented with10% foetal bovine serum, penicillin (100 IU/ml) and streptomycin (100μg/ml). CCL-75 was cultured in MEM with Earle's BSS containing 2 mML-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids,1.5 g/L sodium bicarbonate and 10% foetal bovine serum. LP-9 wascultured in M199 containing 15% CS plus 10 ng/ml of EGF plus 0.4 μg/mlof HC. Saos-2 was cultured in McCoy's 5a medium supplemented with 2 mML-Glutamine and 15% foetal bovine serum. Normal human small airwayepithelial cells (SAEC) and bronchial epithelial cells (BEC) wereobtained from Clonetics (Walkersville, Md.) and cultured in CloneticsSAGM™ Bullet Kit. All cells were cultured at 37° C. in a humid incubatorwith 5% CO₂.

[0139] Antibody Incubation with Cells

[0140] Cells were plated in 6-well plates one day before experiments.Then normal media were replaced by media containing antibodies atvarious concentrations and the cells were incubated at 37° C. in a humidincubator with 5% CO₂. At various time points the cells were collectedusing standard protocols for further analysis. Purified anti-Wnt-1 andanti-Wnt-2 polyclonal antibodies (IgG from goat) were obtained fromSanta Cruz Biotechnology (Santa Cruz, Calif.). As a control, purifiedanti-SOCS-3 (SOCS-3 is a cytoplasmic protein) polyclonal antibody (IgGfrom goat) (also from Santa Cruz Biotechnology (Santa Cruz, Calif.)) wasused in parallel experiments.

[0141] Western Blotting

[0142] Standard protocol as described previously (Yoshikawa et al., NatGenet, 28:29-35 (2001)) was used. Anti-Dvl3, anti-survivin, andanti-Bc1-2 antibodies were obtained from Santa Cruz Biotechnology (SantaCruz, Calif.). Anti-caspase3, anti-caspase9 antibodies were fromOncogene (Cambridge, Mass.). Anti-β-actin, anti-Smac/Diablo andanti-β-catenin antibodies were obtained from Cell Signaling Technology,Inc. (Beverly, Mass.). Anti-cytochrome c antibody was obtained from BDBiosciences. Anti-Active®-JNK antibody was obtained from Promega(Madison, Wis.). For detecting alteration of β-catenin cytosolicextracts were prepared and examined as described previously (Wang etal., Mol Cell Biol, 19:5923-5929 (1999)).

[0143] Apoptosis Analysis

[0144] Cells were harvested by trypsinization and stained using anAnnexin V FITC Apoptosis Detection Kit (Oncogene, Cambridge, Mass.),according to the manufacture's protocol. Then stained cells wereimmediately analyzed by flow cytometry (FACScan; Decton Dickinson,Franklin Lake, N.J.). Early apoptotic cells with exposedphosphatidylserine but intact cell membranes bound to Annexin V-FITC butexcluded propidium iodide. Cells in necrotic or late apoptotic stageswere labeled with both Annexin V-FITC and propidium iodide.

[0145] RNA Interference Analysis

[0146] Cells were plated into a 6-well plate with fresh medium withoutantibiotics 24 hrs before experiments. The ion-exchange HPLC-purifiedsiRNAs (Wnt-1 siRNA and nonsilencing siRNA control, >97% pure) werepurchased from Qiagen-Xeragon (Germantown, Md.). The lyophilized siRNAswere dissolved in annealing buffer and reheated to 95° C. for 1 minfollowed by 1 hr at 37° C. incubation. The siRNA analysis was performedas previously described protocol (Elbashir, et al., Methods 26, 199-213,2002) with some modifications. After siRNA transfection, plates wereincubated for 3-5 days at 37° C. before further analysis.

[0147] In Vivo Tumor Suppression Study

[0148] Human NSCLC cell line H460 and human breast cancer cell lineMCF-7 were cultured as described in previous section. Female nude mice,5-10 weeks old, were injected with 4×10⁶ tumor cells in the dorsal areain a volume of 100 μl. Animals were then intraperitioneally injectedwith monoclonal anti-Wnt-1 antibody, a control monoclonal antibody, orPBS buffer in a volume of 100 μl as well. Both the monoclonal anti-Wnt-1antibody and the control monoclonal antibody were injected at the doseof 50 μg. Each injection was done once weekly. Each group consisted of 5mice. Tumor size was determined at weekly intervals according tostandard techniques.

[0149] Statistical Analysis

[0150] Data shown represent mean values (±S.E.M.). Unpaired T-Test inthe Excel was used for comparing different treatments and cell lines.

[0151] Results

Example 1 Anti-Wnt Antibody Specifically Induces Apoptosis in a Numberof Different Human Cancer Cells

[0152] We examined whether neutralizing Wnt signaling by using anti-Wntantibodies could inhibit cell survival in these cancers. When weincubated a number of cancer cell lines with either anti-Wnt-1 or Wnt-2antibody (at 10 μg/ml) for about 32 hrs (we examined threenon-small-cell lung cancer (NSCLC), two breast cancer, two colorectalcancer, one sarcoma, and two mesothelioma cell lines), we found thatboth antibodies could cause significant cell death (from 30% to 97%),except for one colorectal cancer cell line SW480 (only 4-8%) (FIG. 1).In contrast, an antibody against a cytoplasmic protein (SOCS3) (at.10μg/ml) did not show dramatic cytotoxicity in most of those cell lines(from 4% to 45%) (FIG. 1). Interestingly, none of the antibodies haddramatic effect on the two normal cell lines that we examined (one wasnormal lung fibroblast (CCL-75) and the other was normal mesothelialcell line (LP-9)) (from 2% to 8%) (FIG. 1).

[0153] To determine whether anti-Wnt antibody mediated cell death wasdue to modification of apoptosis, the cells were stained with AnnexinV-FITC and propidium iodide (PI) after antibody treatment for about 32hrs, followed by apoptosis analysis using flow cytometry. As shown inFIG. 2, we found that in the cancer cell lines we examined majority ofcell death was via apoptosis (from 28% to 91%). Again, apoptosis was notdetected in the two normal cell lines after the antibody incubation(only 2% to 6%) (FIG. 2). These results demonstrated that blocking Wntsignaling by using anti-Wnt antibody could specifically induce apoptosisin cancer cells, but not in normal cells.

Example 2 Anti-Wnt Antibody-Induced Apoptosis is Correlated with the WntExpression

[0154] To investigate whether anti-Wnt antibody-induced apoptotic effectwas associated with status of the Wnt proteins, we examined e Wntexpression in the cell lines we tested. As shown in FIG. 3A, we foundthat Wnt-1 had high-level expression in the cancer cell lines that weresensitive to anti-Wnt-1 antibody treatments. However, in the normal lungcell line CCL-75 that was not sensitive to the antibody treatment (seeFIG. 1) only minimal Wnt-1 and expression was detected. No Wnt-1expression was detected in two primary normal lung cells (small airwayepithelial cells (SAEC) and bronchial epithelial cells (BEC)) (FIG. 3)and in normal mesothelial cell line (LP-9) (data not shown). Similarobservations were made regarding Wnt-2 expression.

[0155] As a control, we examined apoptosis induction of co-incubation ofanti-Wnt antibody and blocking peptide for anti-Wnt antibody in an NSCLCcell line. After about 24 hr incubation we found that anti-Wnt antibodyinduced apoptosis could be inhibited by its blocking peptidesignificantly (P<0.01). Taken together, these results indicated thatanti-Wnt antibody-induced apoptosis was correlated with the Wntexpression in the cells we examined.

Example 3 Anti-Wnt-1 Antibody-Induced Apoptosis is a Fast Process andDose Dependent

[0156] We performed dosage and time course experiments on two NSCLC celllines: H838 and A549 (FIG. 4A and FIG. 4B). Flow cytometry analysisafter about 32 hr incubation of anti-Wnt antibody showed that 1 μg/mlantibody could induce apoptosis. A concentration of 20 μg/ml of eitherantibody caused dramatic apoptotic cell death. Anti-Wnt-1 antibody (atconcentration of 8 μg/ml) induced apoptosis could be detected as earlyas after 6 hr incubation and after 50 hr incubation almost all cellswere found undergoing apoptosis or necrosis. In contrast, controlanti-SOCS3 antibody did not have effect on those cancer cells in theparallel experiments. Anti-Wnt-1 antibody incubation with normal lungcell line (CCL-75) was also insensitive to either time or dosage.

Example 4 Anti-Wnt Antibody-Induced Apoptosis is Associated withDown-Regulation of Dvl-3 and Cytosolic β-catenin

[0157] Wnt signaling has been shown to activate β-catenin/Tcf-mediatedtranscription through Dvl. Wnt signaling also stabilizes cytosolicβ-catenin. Thus, we determined whether anti-Wnt antibody inducedapoptosis was dependent on Dvl and destabilization of cytosolicβ-catenin. We found that both Dvl and cytosolic β-catenin level wasdramatically down regulated after anti-Wnt antibody treatment in thecancer cells we examined. In contrast, no change of both Dvl andcytosolic β-catenin level was found in the normal cell line afteranti-Wnt antibody treatment. We also detected apoptosis after we treatedcancer cells with Apigenin that blocks CK-1 activity, which in turninhibits Dvl activity. The cytosolic β-catenin level was downregulatedby Apigenin treatment. These results suggested that anti-Wnt antibodyinduced apoptosis was, at least in part, through inhibiting the functionof Dvl/β-catenin, the downstream components of the Wnt/Frizzledsignaling pathway.

Example 5 Anti-Wnt Antibody Induces Apoptosis Through Down-Regulation ofSurvivin Expression and Subsequent Activation of Caspase-3

[0158] Next, we examined the molecular mechanism of this specificanti-Wnt antibody-induced apoptosis in cancer cells. It has been foundthat activating caspase-9 switches on apoptotic pathway and activatedcaspase-9 amplifies the apoptotic pathway by cleaving and activatingdown stream executive caspases, such as caspase-3. Survivin (one of theapoptosis inhibitor IAP family members) plays an important role ininhibiting activation of both caspase-3 and caspase-9. In cancer cellsthat were sensitive to anti-Wnt antibody treatment both cleaved (active)form of caspase-9 and caspase-3 were up regulated. We also found thatsurvivin expression was significantly down regulated in these cancercells. In contrast, in the normal cell line CCL-75 that was notsensitive to anti-Wnt antibody treatment we did not detect up regulationof cleaved form of both caspases and down regulation of survivinexpression. These results demonstrated that anti-Wnt antibody inducedapoptosis by inhibiting apoptosis inhibitor-survivin and activating ofcaspase-9 and caspase-3.

Example 6 Anti-Wnt Antibody-Induced Apoptosis is Associated withReleasing of Smac/Diablo and Cytochrome c from Mitochondria to theCytosol and JNK Activation

[0159] During apoptosis, Smac/Diablo (second mitochondria-derivedactivator of caspase/direct IAP-binding protein with low pI) functionsto remove the IAP-mediated caspase inhibition. Stimulation of apoptosiscauses releasing of Smac/Diablo from the intermembrane space ofmitochondria into the cytosol, together with cytochrome c. Cytochrome cdirectly activates Apaf-1 and caspase-9 and Smac/Diablo interacts withmultiple IAPs to remove IAP-mediated inhibition of both initiator andeffector caspases. Consistent with above results where caspase-3activity increases in the cancer cells, but not in the normal cells, wefound increase level of both Smac/Diablo and cytochrome c in the cytosolof the cancer cells after anti-Wnt antibody treatment, but not in thatof the normal cells. Our results indicate that both Smac/Diablo andcytochrome c are likely involved in this anti-Wnt antibody inducedapoptosis by removing survivin and/or other IAPs-mediated inhibition anddirect activation of caspases, respectively.

[0160] To further determine how this specific anti-Wnt antibody-inducedapoptosis is regulated, we examined other components in the apoptoticpathway. Surprisingly, we found that JNK activity was dramaticallyincreased in the cancer cells after the treatments. In contrast, in thenormal cell line CCL-75 that was not sensitive to anti-Wnt antibodytreatment increase of JNK activity was not detected. We also found thatover-expression of Dvl in a normal mesothelial cell line down regulatedJNK activities. In addition, inhibition of Dvl by using Apigenin toblock CK-1 activity could also increase JNK activity. Taken together,the anti-Wnt antibody-induced apoptosis involves JNK activation andincrease of the JNK activity after blocking Wnt signaling is likelythrough inactivating Dvl.

Example 7 Anti-Wnt-1 antibody Specifically Induces Apoptosis in Wnt-1Transfected Mouse Mammary Cells

[0161] As one control, we compared apoptotic effect induced byanti-Wnt-1 antibody incubation in mouse C57MG versus Wnt-1-transfectedC57MG cells, because these cells have already been characterized fortheir free pool of β-catenin. It has been shown that Wnt-1 signaling ison in Wnt-1-transfected C57MG cells, but off in un-transfected orempty-vector-transfected C57GM cells. Flow cytometry analysis after 42hr anti-Wnt-1 antibody incubation showed no noticeable effect inun-transfected or empty-vector-transfected C57GM cells (less than 10%cell death after incubation). However, significant cell death was seenin Wnt-1-transfected C57MG cells (over 85% cell death, P<0.001).

[0162] The anti-Wnt-1-induced apoptosis in Wnt-1-transfected C57MG cellsalso appears to be linked with down-regulation of Dvl-3 and cytosolicβ-catenin, and through down-regulation of survivin expression andsubsequent activation of caspase-3, and through releasing of Smac/Diabloand cytochrome c from mitochondria to the cytosol and JNK activation.The Wnt-1-transfected C57MG cell line serves as an ideal control modelfor our discovery, and these data provided more support to our findingin human cancer cells.

Example 8 An anti-Wnt-1 Monoclonal Antibody Shows Induction of Apoptosisin Different Human Cancer Cells in Vitro and Suppresses Tumor Growth inVivo

[0163] Antibodies were raised against peptides derived from human Wnt-1.In particular, hybridoma cell liens were generated using SEQ ID NO:2 andSEQ ID NO:4. One of the monoclonal antibodies was raised against asynthetic peptide corresponding to amino acid 201-212 of the human Wnt-1(Ac-HNNEAGRTTVFS-amide). The antibody was affinity purified usingProtein A. Wnt-1 expression in numerous human cell lines was evaluatedusing this monoclonal antibody. The cell lines included three breastcancer cell lines (HuL100, MCF-7, and SKBR-3), five malignant pluralmesothelioma cell lines (REN, H513, H290, MS-1, and H28), fournon-small-cell lung cancer (NSCLC) cell lines (A549, H460, H838, andH1703), two sarcoma cell lines (MES-SA and Saos-2), one colon cancercell line SW480, and four normal cells (small airway epithelial cells(SAEC) and normal human bronchial epithelial cells (NHBE), LP-9, andCCL-75). We found higher-level Wnt-1 expression in most of these cancercell lines, except for A549, MES-SA, H513, SKBR-3 and SW480, which hadno or minimal Wnt-I expression. No Wnt-1 expression was observed in thetwo primary normal lung cells (SAEC and NHBE). We only detected minimalWnt-1 expression in the normal lung fibroblast CCL-75 and in a normalmesothelial cell line (LP-9). As a control experiment, we found Wnt-1expression using the same monoclonal antibody in Wnt-1-transfected mousemammary cells (C57Wnt-1), but not in empty-vector-transfected cells(C57mv7).

[0164] To test if the anti-Wnt-1 monoclonal antibody can specificallybind to the native form of Wnt-1 protein in cultured cells, we performedimmunoprecipitation using monoclonal antibody alone or monoclonalantibody blocked by pre-incubation with blocking peptide (30-fold overthe antibody) in cell extracts from several cell lines. C57Wnt-1 andC57mv7 cells served as positive and negative controls, respectively.NSCLC (H460) and breast cancer (MCF-7) cell lines were also tested. InC57Wnt-1, H460 and MCF-7 cells Wnt-1 protein was precipitated by theanti-Wnt-1 monoclonal antibody. In contrast, when the anti-Wnt-1monoclonal antibody was preincubated with blocking peptide, its abilityto precipitate Wnt-1 protein was blocked in these cells. No Wnt-1protein was precipitated by either anti-Wnt-1 monoclonal antibody aloneor monoclonal antibody pre-incubated with blocking peptide in thenegative control. These data indicate that the anti-Wnt-1 monoclonalantibody specifically binds to native form of Wnt-1 protein.

[0165] Next, we treated a NSCLC cell line H460 and a breast cancer cellline MCF-7 with this monoclonal antibody. After about 48-72 hrs ofincubation we found significant cell death in both cell lines (over 60%cell death at 10 μg/ml of the antibody, P<0.001) (FIG. 5a). We saw nonoticeable effect, however, in both cell lines after control monoclonalantibody treatment. Cell killing was largely due to induction ofapoptosis (FIG. 5b). Induction of apoptosis by this monoclonal antibodywas dosage and time dependent (over 60% cell death in H460 at 10 μg/mlof the antibody after about 72 hrs of incubation and over 40% cell deathin MCF-7 at 10 μg/ml of the antibody after about 48 hrs of incubation)(FIG. 5c). We also treated other cancer cell lines that have Wnt-1overexpression, including breast cancer HuL100, NSCLC H1703,mesothelioma H28 and REN, and sarcoma Saos-2. We found similar results.

[0166] As a specificity control, we examined induction of apoptosis byusing monoclonal antibody blocked by overnight pre-incubation withblocking peptide (30-fold over the antibody) in H460, MCF-7 and H1703.After 48 hrs of incubation, we found that anti-Wnt-1 antibody-inducedapoptosis could be inhibited significantly by its blocking peptide(P<0.003). Same dose blocking peptide alone did not affect viability ofthese cells (8.0 μg/ml for 48 hrs). As a negative control, we used A549cells that lack significant Wnt-1 expression. After about 48 hrtreatment with either monoclonal antibody alone (8.0 μg/ml) or withmonoclonal antibody blocked by preincubation with blocking peptide(30-fold over the antibody), no significant induction of apoptosis wasdetected. This result is consistent with Wnt-1 expression status of A549cells.

[0167] The Anti-Wnt-1 Monoclonal Antibody Inhibits Wnt/β-cateninSignaling Pathway and Induces Apoptosis Through Release of Cytochrome c,Down-Regulation of Survivin Expression and Subsequent Activation ofCaspase-3

[0168] We found that both Dvl-3 and cytosolic β-catenin as well asCyclin D1 levels were down-regulated after anti-Wnt-1 monoclonalantibody treatment in the cancer cells examined. We also performedTOP/FOP assay in these cells and found that TCF dependenttranscriptional activity decreased after the monoclonal antibodytreatment. In contrast, no change of either Dvl, cytosolic β-Cateninlevels or TCF dependent transcriptional activity was found in normalcells or cancer cells lacking (or with minimal) Wnt-1 expression afteranti-Wnt-1 monoclonal antibody treatment. These results suggest thatanti-Wnt-1 monoclonal antibody induced apoptosis is mediated, at leastin part, through inhibiting Dvl/β-catenin dependent transcription.

[0169] In H460 cells in which anti-Wnt-1 monoclonal antibody inducesapoptosis, we found that cleaved (active) form of caspase-3 wasup-regulated. Consistent with the caspase-3 activity, we detectedincreased level of Cytochrome c in the cytosol of H460 cells afteranti-Wnt-1 monoclonal antibody treatment. In addition, we found thatSurvivin expression was down-regulated in these H460 cells after theantibody treatment.

[0170] Others have shown that Wnt-1 signaling is on in C57Wnt-1 cells,but off in C57mv7cells 11. As a control, we tested if anti-Wnt-1monoclonal antibody could inhibit Wnt/β-catenin signaling in C57Wnt-1cells. Western analysis C57mv7 showed that both cytosolic β-catenin andCyclin D1 levels were down-regulated after anti-Wnt-1 monoclonalantibody treatment (8.0 μg/ml for 48 hrs) in C57Wnt-1 cells, but noCyclin D1 expression was detected in C57mv7 cells. Cytosolic β-cateninlevel in C57mv7 cells also remained unchanged after anti-Wnt-1monoclonal antibody treatment. Consistently, TCF-dependenttranscriptional activity measured by TOP/FOP assay also decreased inC57Wnt-1 cells, but remained unchanged in C57mv7 cells. These dataindicate that the anti-Wnt-1 monoclonal antibody inhibits Wnt/β-cateninsignaling in the cell lines examined.

[0171] RNA Interference

[0172] We followed the protocol described by Elbashir et al. (Elbashir,et al., Methods 26, 199-213, 2002) to investigate the effect ofsilencing Wnt-1 expression by using RNAi. Similar to the monoclonalanti-Wnt-1 antibody, treatment with Wnt-1 siRNA for 3-5 days inducedapoptosis in cancer cell lines, e.g., MCF-7 cells, that express Wnt-1.Significant apoptosis was induced at 100 nM Wnt-1 siRNA, but noapoptosis was induced by either non-silencing siRNA control (100 nM) ortransfection reagents. We confirmed the silencing of Wnt-1 expressionafter Wnt-1 siRNA treatments (100 nM for 72 hrs) by Western analysis(non-silencing siRNA served as control (100 nM for 72 hrs)). Todetermine whether the apoptotic effects correlated with the inhibitionof Wnt-1 signaling, we also showed that expression levels of Dvl-3,cytosolic β-catenin, and Survivin were down-regulated after Wnt-1 siRNAtreatment.

[0173] Inhibition of Cancer Growth in Vivo

[0174] Next we tested whether the monoclonal anti-Wnt-1 antibody couldsuppress tumor growth in vivo. We injected H460 and MCF-7 cells intonude mice, respectively. Animals were then received 50 μg of themonoclonal anti-Wnt-1 antibody, a control monoclonal antibody or PBS viaintraperitoneal (i.p.) injection once weekly. FIG. 6A shows although thecontrol antibody had no appreciable suppression, the monoclonalanti-Wnt-1 antibody at such dose significantly inhibited growth of bothtumor types (P<0.001). Suppression of the tumor growth was seen not onlywhen the monoclonal anti-Wnt-1 antibody injection was startedimmediately after tumor cell inoculation (FIG. 6A), but also when thetreatment was initiated after the tumors were already established (oneweek after tumor cell inoculation) (P<0.005) (FIG. 6B). In the studiesusing MCF-7 cells (FIG. 6C), tumor volume is whons after 3 weekstreatment with anti-Wnt-1 monoclonal antibody and control monoclonalantibody. Five animals are in each group. None of the animals that weretreated with anti-Wnt-1 mAb injections developed tumors. However, threeof five control animals developed tumors. (I.P. injections wereadministered once weekly one week after MCF-7 cell inoculation.).

[0175] The sequences of the V_(H) and VL regions of the anti-Wnt-1monoclonal antibody used in the studies described above were determined.The CDR and framework (FR) regions were amplified from the hybridomacell lines by RT-PCR and analyzed by agarose gel. The sequences of theV_(H) and VL regions are shown in FIG. 7.

Example 9 Wnt-2 Expression and Wnt-2 Monoclonal Antibody-InducedApoptosis.

[0176] Wnt-2 gene expression was analyzed in multiple human cancer andmatched non-cancerous tissue specimens. Radiolabeled Wnt-2 cDNA probeswere hybridized with the Cancer Profiling Array II (BD Biosciences,Inc.), which contains 19 different types of human tumors with matchednon-cancerous tissue specimens. Wnt-2 was overexpressed in the majorityof colon, stomach, rectal, and thyroid tumors in comparison with theirnormal counterparts.

[0177] A monoclonal antibody was raised against a synthetic peptidecorresponding to amino acids 49-63 (SSQRQLCHRHPDVMR) of human Wnt-2. Theantibody was affinity purified using Protein A. The effect of Wnt-2monoclonal antibodies on apoptosis was determined in human melanoma FEMXand LOX cells. The results show that the anti-Wnt-2 monoclonal antibodyinduced apoptosis in FEMX and LOX human melanoma cells. The antibodyalso induced apoptosis in human colon cancer HCT-116 and SW480 cells, asdid the anti-Wnt-1 monoclonal antibody of Example 8.

[0178] The sequences of the V_(H) and VL regions of the anti-Wnt-1monoclonal antibody used in the studies described above were determined.The CDR and framework (FR) regions were amplified from the hybridomacell lines by RT-PCR and analyzed by agarose gel. The sequences of theV_(H) and V_(L) regions are shown in FIG. 7.

EXAMPLE 10 Mesotheliomas Have Over Expression of β Catenin ThroughActivation of Dvl, and Transcriptional Activity of β Catenin isCorrelated to Tumorigenecity

[0179] We further investigated the role-of wnt signaling inmesotheliomas. We found that most mesothelioma cells overexpress Dvl-3.Expression of Dvl-3 and cytosolic β-Catenin was investigated inmesothelioma cells using western blots. Western blot analysis showedthat 8 of 10 fresh malignant mesothelioma tissues overexpress Dvl-3protein and have increased cytosolic β-catenin compared with autologousmatched normal pleural tissue controls. Furthermore, five additionalmalignant mesothelioma cells tested (two primary malignant pleuralmesothelioma cultured cells and three cell lines, LRK1A, REN, and H513)had high levels of Dvl-3 and cytosolic β-catenin, compared with normalpleuralbcontrols. Immunohistochemical analysis of several of the tumorcells demonstrated cytoplasmic, nuclear, and membrane bound β-catenin.We found no mutation in exon 3 of β-catenin in 13 mesothelioma tissues,including the cases tested by Western blot and two malignant effusions.Exon 3 was selected for mutational analysis because it encodes theNH2-terminal regulatory domain of β-catenin, which was previously foundto contain activating mutations. Furthermore, we detected no mutation inthe complete coding region of β-catenin in three mesothelioma cell lines(LRK1A, REN, and H513).

[0180] Transcription activity of β-catenin using a Tcf-dependentluciferase reporter gene was also examined. Western blot analysis wasused to confirm APC, GSK-3β, and Tcf4 expression in all tumors understudied. Transcriptional activity mediated by Tcf-β-catenin proteincomplexes was assayed as a ratio to reporter gene activity inmesothelioma cell lines with significant overexpression of Dvl andcytosolic β-catenin. Cells were transiently transfected with either thepTOPFLASH or pFOPFLASH reporter construct, which contained multimerizedwild type or mutant Tcf-binding motifs upstream of the FireflyLuciferase cDNA with the pRL-TK internal control reporter construct thatcontains the Renilla Luciferase cDNA. Tcf-mediated gene transcriptionwas determined by the ratio of pTOPFLSH:pFOPFLLASH luciferase activityafter 24 h, each corrected for luciferase activities of the pRL-TKreporter.

[0181] Mesothelioma cells with high levels of cytosolic β-catenin,including cells from malignant pleural mesothelioma effusions, LRK1A,REN, and H513 cell lines showed a significant fold increase(1.5-2.4-fold, P<0.01) in Tcf-mediated gene transcriptional activity ofP-catenin (pTOPFLASH/pFOPFLASH). In contrast, normal mesothelial cells,which have minimal expression of cytosolic β-catenin, showed nodifference.

[0182] Transcriptional activity of β-catenin in Tcf β-catenin mediatedreporter assay, which was confirmed by the reporter assay underexpression of Gal4-β-catenin fusion protein, categorized mesotheliomacells, which was positive transcriptional activity or negative. Inmesothelioma cells, the cytosolic expression of β-catenin was inhibitedby adding apigenin, which can degradate Dvl through corruption of caseinkinase II, and PDZ-Dvl, but was enhanced by wild type Dvl. Furthermore,PDZ-Dvl inhibited the Tcf dependent transcriptional activity. Stablyexpression of PDZ-DVL inhibited the colony formation in the mesotheliomacells, which had the positive transcriptional activity of β-catenin, butthe mesothelioma cells, which had negative transcriptional activity ofβ-catenin, showed satble colony formation. Our confirmation of Tcfdependent transcription and Gal4-β-catenin fusion protein was wellcorrelated to the results of the tumorigenesity in mesothelioma cell.

[0183] Dvl-3 stabilizes the cytosolic β-catenin in mesothelioma cells.We have confirmed that fresh malignant mesothelioma cells from pleuraleffusions demonstrated the overexpression of Dvl-3 protein withexpression of the cytosolic fi-catenin by Western blot analysis. Exceptfor H28 cell line, which contains a homozygous deletion of β-cateninregion, all other malignant cells tested with high expression of Dvl-3showed remarkably higher expression of cytosolic β-catenin than cellsfrom normal pleural tissue. These results demonstrate that activation ofDvl-3 translocate β-catenin from membrane to cytoplasm and nucleus inmesothelioma cells.

[0184] β-catenin activates the Tcf dependent transcription inmesothelioma cells. Transcriptional activation mediated by Tcf-β-cateninprotein complexes was determined and compared by reporter gene analysisin mesothelioma cell lines with significant overexpression of Dvl andcytosolic β-catenin, and another mesothelioma cell line, H28, that lacksexpression of β-catenin due to homozygous deletion but contains theexpression of Dvl. Cells were transiently transfected with either thepTOPFLASH or pFOPFLASH reporter construct, which contained multimerizedwild type or mutant Tcf-binding motifs upstream of the FireflyLuciferase cDNA with the pRL-TK internal control reporter construct thatcontains the Renilla Luciferase cDNA. Tcf-mediated gene transcriptionwas determined by the ratio of pTOPFLSH:pFOPFLLASH luciferase activityafter 24 h, each corrected for luciferase activities of the pRL-TKreporter. Of mesothelioma cells with high expression of cytosolicβ-catenin, malignant effusion of a mesothelioma patient, LRK1A and RENshowed 1.8-2.4 fold increase in transcriptional activity of thepTOPFLASH reporter, and H290 and H513 exhibited 1.4-1.5 fold increase.In contrast, H28 and normal mesothelial cells, which have no or slightexpression of cytosolic β-catenin, showed no deference between thepTOPFLASH and pFOPFLASH activity. These results indicate that a greatdeal of β-catenin can be the transmitter in mesothelioma cells.

[0185] Gal4-β-catenin activates pG5 in mesothelioma cells. Furthermore,the control of transcriptional activity by β catenin in mesotheliomacells was measured using the GAL4-β-catenin construct to exclude thepossibility that mesothelioma cell lines lack the necessarytranscriptional machinery. After cotransfection ofpSG424-GAL4-β-catenin, which transcribes the GAL4-β catenin fusionprotein, and pG5, a CAT reporter construct, GAL4-β-catenin mediated genetranscription was determined. These activities were normalized to theCAT activity of the pG5 reporter construct only to exclude thebackground level of activation. Gal4-β-catenin protein was expressed inall transfected mesotheliomas by Western blot analysis using Flagantibody. LRK1A, REN and H28 cells showed 10-25-fold increased activityafter co-transfection with pSG-GAL4-β-catenin and pG5 reporter constructas compared with control transfection of pG5. Hela cells exhibited a25-fold increase. In contrast, H513 showed a few-fold increase, and H290showed only background activity. These high activity confirm that theabundant β-catenin mesotheliomas is capable of transcriptional activityin LRK1A and REN, but it is impossible in H290, even though it has ahiger activation in Tcf dependent transcription.

[0186] Apigenin induces the degradation of Dvl, which results in thestability of cytosolic β-catenin. Apigenin promotes degradation of Dvland β-catenin through inhibition of casein kinase II in mammaryepithelial cells, leading to the inhibition of cell proliferation.Adding Apigenin to media inhibited the growth of LRK1A, REN and H290over the course of a 48 hours treatment degradated Dvl and cytosolicβ-catenin. These results suggest that the activation of Dvl by caseinkinase II regulates, in part, the translocation of β-catenin inmesothelioma cells.

[0187] PDZ-Dvl inhibits the function of endogenous Dvl and the stabilityof cytosolic β-catenin in mesothelioma cells. Dishevelled proteinspossess three conserved domains, a dix domain, present in the Wntantagonizing protein Axin; a PDZ domain involved in protein-proteininteractions, and a DEP domain found in proteins that regulate RhoGTPases. Function of three conserved domains is required forup-regulation of β-catenin and for stimulation of LEF-1-mediatedtranscription in mammalian cells. Transfection of pCS-mouse Dvl-1 to293T cells resulted in a 15-fold increase in Tcf-mediated genetranscriptional activity of β-catenin, in accordance with otherinvestigators' findings. This activity was inhibited by a pCS-mouseDvl-1 construct by cotransfection of pCS-cDNA-encoding APDZDvl-1.Furthermore, Tcf-dependent transcriptional activity of β-cateninbinLRK1A was reduced by transfection of pCS-ΔPDZ-Dvl-1 (from 2.1- to1.3-fold, P<0.05), whereas transfection of pCS-Dvl-1 enhancedTcf-dependent transcriptional activity of β-catenin (from 2.1- to3.8-fold, P<0.05), indicating that β-catenin Tcf-mediatedbtranscriptionin these cells is regulated significantly by Dvl.b.

[0188] To examine additional Wnt pathway activation in malignant pleuralmesothelioma, we transfected retrovirally, ΔPDZ-Dvl-1 and wild-typeDvl-1 into LRK1A, REN, and H513 cell lines, respectively. Retrovirustransfection of pLXN-ΔPDZ-Dvl-1 induced expression of ΔPDZ-Dvl-1protein, which significantly reduced the expression of cytosolicβ-catenin in all cells tested compared with controls (P<0.05). Theseresults demonstrate that Dvl regulates cytosolic β-catenin inmesothelioma cells.

[0189] Using Atlas human cancer 1.2 array, c-myc expression in REN wasshown to be down-regulated by ΔPDZ-Dvl-1 transfection. On the otherhand, COX-2, which has been confirmed to be one of target genes ofWnt/β-catenin pathway, was down-regulated by APDZ-Dvl-1 transfectionusing Western blot analysis.

[0190] Transfection of ΔPDZ-Dvl Inhibits Tumorigenicity of MesotheliomaCell Lines in Soft Agar and in Athymic Mice.

[0191] We examined the role of the Dvl/β-catenin pathway in relationshipto cell growth in malignant pleural mesothelioma cell lines. We inducedexpression of ΔPDZ-Dvl-1 in LRK1A, REN, and H513 through retroviraltransfection, using empty vector as a control. After selection, cellswere plated in 0.35% soft agar and colonies scored after 28 days. Colonyformation in LRK1A and REN transfected with ΔPDZ-Dvl-1 decreasedsubstantially compared with control (P<0.01). H513 was unable to grow insoft agar. In addition, the in vivo growth of both LRK1A and REN s.c.tumors in athymic mice was inhibited significantly by transfection witha ΔPDZ-Dvl-1 mutant compared with control (P<0.05 and P<0.005,respectively; FIG. 8).

Example 11 Role of Dvl Activation in Non Small Cell Lung Cancer

[0192] We next examined the role of Dvl activation in non small-celllung cancer (NSCLC). This example demonstrates that Dvl-3 isoverexpressed in freshly resected NSCLC and established NSCLC celllines. We example also provides additional evidence that Wnt signalingthrough canonical β-catenin pathways is due to upstream events, such asDvl expression.

[0193] We analyzed Dvl expression and function in order to evaluate thefunction of wnt signaling in NSCLC. Eight NSCLC fresh tumors (foursquamous cell and four adenocarcinomas) and their autologous matchednormal lung tissue were obtained from patients undergoing resection oftheir tumors as part of their treatment for early stage I NSCLC.Patients had not received any prior treatment, e.g., chemotherapy.Western blot analysis of these samples showed that in 75% (three of foursquamous cell carcinomas and three of four adenocarcinomas) of allcancer cells tested, Dvl-3 was overexpressed while the correspondingmatched normal microdissected lung tissues failed to show expression ofDvl-3. Furthermore, five of six NSCLC tumors with Dvl-3 over-expressionshowed higher expression of Wnt-1 or Wnt-2 by western blot analysis.Expression of Dvl-1 or Dvl-2 was not detected.

[0194] To further examine Dvl function, we synthesized small interferingRNA (siRNA) of Dvls that are capable of suppressing Dvl-1, -2, and -3.We tested the function of Dvl in the lung cancer cell line H1703 bytreatment with Dvl siRNA and control siRNA. We chose H 1703 because itexpresses Dvl-3 and has been shown to exhibit Tcf-dependenttranscriptional activity of β-catenin. After siRNA treatment, expressionof dvl-3 was suppressed, while dvl-1 and -2 remained unexpressed. Ofnote, β-catenin expression decreased accordingly in treated cells, whichwas accompanied by a significant reduction in Tcf-dependenttranscriptional activity (P<0.05). Lastly, siRNA of Dvls inhibited H1703cell growth in 24-well plates significantly (P<0.05) (FIG. 9). Inaddition, colony formation in 100-mm dishes was also suppressedsignificantly (P<0.05). In other cell lines with lower levels of Dvlexpression compared to that in H1703, such as A549 (a lung cancer cellline) and SW480 (a colon cancer cell line with aberrant activation inthe Wnt signaling pathway due to APC mutation), cell growth wasunaffected by the Dvl siRNA.

[0195] Discussion

[0196] As noted above, little is known regarding the role that wntligand plays in human carcinogenesis. The data presented heredemonstrate that wnt signals play a causal role in human cancer cellsand thus are cancer therapeutic targets.

[0197] The data presented above demonstrate that both anti-wnt-1 andanti-wnt-2 antibodies can induce apoptosis in human cancer cells.Furthermore, our data indicates that the anti-tumor effect was due tothe blockade of wnt signaling pathway. The apoptotic cell death inducedby anti-Wnt antibody was not only correlated with the Wnt proteinexpression, but also consistent with the decreased dvl and cytosolic βcatenin protein expression in the human tumor cells tested. Conversely,both Dvl and cytosolic β-catenin proteins remain the same level innormal cell lines after anti-Wnt antibody treatment. The antibodiesshowed no detectable effect on normal cell lines, suggesting thatanti-Wnt-1 or anti-Wnt-2 antibody could specifically induce apoptosis incancer cells, but not in normal cells. Given the possibility thatpolyclonal antibodies may generate non-specific effects, we used ananti-wnt-1 monoclonal antibody to further investigate the specificity ofthe effect of anti-wnt antibodies. The anti-wnt-1 monoclonal antibodywas able to induce apoptosis in human cancer cell lines thatover-express Wnt-1 protein, e.g., human lung cancer cell line H460 andhuman breast cancer cell line MCF-7. Similar to the results obtainedfrom polyclonal antibody study, both dvl and cytosolic β cateninproteins were decreased after the anti-Wnt-1 monoclonal antibodytreatment in these tumor cells. However, the anti-Wnt-1 monoclonalantibody showed much higher specificity than the anti-Wnt-1 polyclonalantibody, e.g., the anti-Wnt-1 monoclonal induces apoptosis only in thetumor cells that over-express Wnt-1 protein (H460 and MCF-7), and has nodetectable effect in the tumor cells that express Wnt-2 protein; theanti-Wnt-1 polyclonal antibody induces apoptotic cell death in the tumorcells that over-express either Wnt-1 or Wnt-2. Taken together, thesedata indicate that the anti-Wnt antibody treatment can inducetumor-specific apoptosis and down-regulate the Wnt-dvl-β cateninsignaling pathway in human cancer cells.

[0198] Through frizzled receptor and dishevelled protein, Wnt signalactivates two distinct pathways: the canonical pathway (i.e., βcateninpathway) and the JNK pathway. Dishevelled protein has three highlyconserved domains, DIX, PDZ, and DEP. Among them, the DIX and PDZdomains are necessary for the canonical signaling pathway while the DEPdomain is important for the activation of JNK pathway. It has beensuggested that the activation of JNK plays a critical role in initiatingapoptosis (Wang et al., Mol Cell Biol, 19:5923-5929 (1999)). Recently,Chen et al. have demonstrated that Wnt-1 inhibits apoptosis byactivating βcatenin and TCF transcription (Chen et al., J Cell Biol,152:87-96 (2001)). In this study, both over-expression of β-catenin andincreased JNK activity were observed after anti-Wnt antibody treatment,suggesting that both the canonical pathway and the JNK pathway areinvolved in the apoptosis induced by anti-Wnt antibody. In addition,over-expression of Dvl in a normal mesothelial cell line down regulatedJNK activities and the inhibition of Dvl by using Apigenin to block CK-1activity increased JNK activity. Most likely, the activation of JNKafter anti-Wnt antibody treatment is through Dvl.

[0199] Furthermore, siRNA-mediated inhibition of Dvl expression in NSCLCcells decreased β-catenin-mediated Tcf transcription, which furthersupports that Dvl overexpression is important to the canonicalWnt/B-catenin pathway in some lung cancer cells. Inhibition of Dvl alsosuppressed cell growth and colony formation in NSCLC cells, whichindicates that aberrant upstream events in Wnt signaling is related totumorigenesis in NSCLC.

[0200] Degradation of Dvl by siRNA resulted in growth suppression inHI703, but not in A549 cells. These are both squamous cell lung cancercell lines, but H1703 has mutational inactivation of p53 whereas A549has wild-type p53. The p53 status may therefore explain, at least inpart, the differences in Dvl function between the two squamous cell lungcancer cell lines treated.

[0201] To further elucidate the mechanism through which anti-Wntantibody induce apoptosis in human cancer cells, we have examined otherpossible components in the apoptotic pathway. For instance, releasing ofSmac/Diablo into cytosol was detected in these tumor cells treated withwnt antibody. Smac/Diablo (second mitochondria-derived activator ofcaspase/direct IAP-binding protein with low pI) (Du et al., Cell,102:33-42 (2000); Verhagen et al., Cell, 102:43-53 (2000)) functions byreleasing the IAP-mediated caspase inhibition. Stimulation of apoptosiscauses releasing of Smac/Diablo from the intermembrane space ofmitochondria into the cytosol, together with cytochrome c. Cytochrome cdirectly activates Apaf-1 and caspase-9 and Smac/Diablo interacts withmultiple IAPs to remove IAP-mediated inhibition of both initiator andeffector caspases (Chai et al., Nature, 406:855-862 (2000); Srinivasulaet al., J Biol Chem, 275:36152-36157 (2000)). Consistent with aboveresults where caspase-3 activity increases in the cancer cells, but notin the normal cells, we found increase level of both Smac/Diablo andcytochrome c in the cytosol of the cancer cells after anti-Wnt antibodytreatment, but not in that of the normal cells. Our results indicatethat both Smac/Diablo and cytochrome c are likely involved in thisanti-Wnt antibody induced apoptosis by removing survivin and/or otherIAPs-mediated inhibition and direct activation of caspases,respectively.

[0202] The above findings suggest that wnt antibodies may not onlyinduce directly apoptosis in cancer cell that overexpress wnt proteins,but also release potentially drug resistance by restoring normalapoptotic machinery back to these tumor cells. The basis for drugresistance in tumor cells is most likely the disruption of apoptosis.Over expression of Survivin, an inhibitor of apoptosis, is a commonfeature of most human cancers. It has been shown that targeting ofsurvivin increases the sensitivity of tumor cells to cytotoxic drugs andthat antisense survivin is sufficient to cause apoptosis in humanmesothelioma cells. Moreover, a synergistic effect between antisensesurviving and chemotherapy has also been reported.

[0203] We have shown that wnt antibody treatment dramatically decreasesthe protein expression level of Survivin. Taken together, Wnt antibodyshould potentiate and synergize the effect of standard chemotherapy inhuman cancer cells.

[0204] Other antagonists of Wnt signal or Frizzled receptor should alsoinduce apoptosis through dishevelled. For instance, sFRPs function assoluble modulators of Wnt signaling by competing with the Frizzledreceptors for the binding of secreted Wnt ligands (Melkonyan et al.,Proc Natl Acad Sci USA, 94:13636-13641 (1997)). Specifically, sFRPs caneither antagonize Wnt function by binding the protein and blockingaccess to its cell surface signaling receptor, or they can enhance Wntactivity by facilitating the presentation of ligand to the Frizzledreceptors (Uthoff et al., Int J Oncol, 19:803-810 (2001)). Frizzledreceptor antagonists (e.g., antibody specific for the extracellulardomain or small molecule specific for the intracellular domain) shouldinduce apoptosis in human cancer cells that overexpress wnt/frizzledproteins. Indeed, FIG. 10 shows that over-expression of Wnt signalantagonist, FRP or DKK, induces apoptosis in cancer cells. Thus, suchantagonists can also be used to treat cancer, e.g., lung cancer,mesothelioma, breast cancer, colorectal cancer, cervical cancer, ovanancancer, prostate cancer, pancreatic cancer, gastric cancer, esophagealcancer, head and neck cancer, hepatocellular carcinoma, melanoma,glioma, glioblastoma, leukemia, or lymphoma.

[0205] In summary, our results indicate that wnt monoclonal antibodiescan induce tumor-specific apoptosis in human cancer cells, probablythrough both the canonical and the JNK pathways. Our data demonstratethat Wnt/Frizzled is a useful therapeutic targets for the treatment ofcancer, and the results from xenograft mouse model implicate that Wntmonoclonal antibodies are good candidates of tumor-targeting cancertherapeutics.

Example 12 Analysis of Silencing Mechanisms of the Dachsous (ds) and FatGenes and their Regulations of the Wnt-Frizzled Signaling Pathway inHuman Cancers

[0206] Little is known regarding modification machinery of Wnt-Fzsignaling in cancers. Dachsous (Ds) and Fat proteins are two cadherinsuperfamily members (Mahoney, et al., Cell 67: 853-868, 1991; Clark, H.F., et al., Genes Dev, 9: 1530-1542, 1995). They have been shown toparticipate in Fz signaling in Drosophila development (Yang, et al.,Cell, 108: 675-688, 2002). There is no report on the role of Ds and Fatin cancers.

[0207] In this example, we show that in fresh human cancer tissues(including lung cancer and mesothelioma) and human cell lines (includingbreast cancer, colon cancer, lung cancer and mesothelioma) Fatexpression was upregulated and Ds expression was downregulated. We alsoidentified aberrant methylation in the CpG island of Ds promoter regionthat correlated with Ds transcription silencing in human cancers. Inaddition, we found that Fz activity was correlated with upregulation ofFat and downregulation of Ds. Restoration of Ds and blockage of Fatcould modulate activity of the Fz signaling pathway and suppress cancercell growth.

[0208] This invention is of great help in therapeutic strategies for thetreatment of human cancers. For example, using the methods describedabove, Fat activity can be blocked. Such methods include, for example,antisense oligonucleotides or small chemical molecules to block Fattranscript and/or its activity. Alternatively, Ds activity can berestored using known gene therapy methods. In addition, this inventioncan be used as a diagnostic tool. Methylation is one of early events incancer formation. Methylation detection in the CpG island of Ds promoterregion using well known techniques, for example, methylation-specificPCR can be used on early cancer diagnosis.

Example 13 Genetic Alterations in Frizzled (fz) Genes and LRP(LDL-Related Protein) Genes and Targeting Mutant and/or Truncated Formsof These Receptors Using Different Methods in Cancers

[0209] LRPs (LDL-related protein; LRP-1 to 5) are co-receptors for Wntligands. It has been shown that Wnt, Fz and LRP proteins often have highlevel expression in a number of cancers, including breast cancer, coloncancer, lung cancer etc. (Liu, et al., Cancer Res, 60: 1961-1967, 2000;Laurencot, et al., Int J Cancer, 72: 1021-1026, 1997; Berger, W., etal., Int J Cancer, 88: 293-300, 2000; Schneider, et al., Breast CancerRes, 3: 183-191, 2001; Schneider, et al., Anticancer Res, 20: 4373-4377,2000). In addition, this signaling is thought to turn on downstreamtranscriptional activity constitutively in cancers. However, mechanismsin this constitutive-on signaling in cancers still remain unsolved.

[0210] In this example, we show that genetic alterations infrizzled (fz)genes and/or LRP (LDL-related protein) genes result in mutant and/ortruncated forms of all Fz receptors and/or LRP co-receptors(extracellular, transmembrane, and/or intracellular domains) in cancers.The cancer types that we tested include breast cancer, colon cancer,prostate cancer, lung cancer, mesothelioma, and sarcoma. The geneticalterations mentioned above include chromosomal deletion (homozygous orheterozygous), chromosomal translocation, chromosomal breaks,chromosomal inversions, internal small deletions, insertions, and pointmutations. These mutant and/or truncated forms of Fz receptors and/orLRP co-receptors result in constitutive signaling regardless presence ofWnt ligands, which in turn result in constitutive downstreamtranscriptional activities in cancers. In contrast, there are no mutantforms of Fz receptors and/or LRP co-receptors in normal cells/tissues.

[0211] This invention demonstrates that mutant and/or truncated forms ofFz receptors and/or LRP co-receptors for the Wnt signaling pathway arecancer specific. They have very strong potential to be used as targetsfor developing therapeutic drugs (e.g., small molecules, chemicalcompounds, antibodies, antisense-oligos or RNAi as discussed above).These drugs are able to target cancers only, but not normal cells. Thus,this invention will be of great help in therapeutic strategies fortreatment of a number of cancers as noted above, including colon cancer,breast cancer, lung cancer, e.g., NSCLC, mesothelioma and sarcoma, andthe like.

[0212] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes. SEQUENCES Seq ID No:1Human Wnt-1 peptide sequence #1MGLWALLPGWVSATLLLALAALPAALAANSSGRWWGIVNVASSTNLLTDSKSLQLVLEPSLQLLSRKQRRLIRQNPGILHSVSGGLQSAVRECKWQFRNRRWNCPTAPGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRRRGPGGPDWHWGGCSDNIDFGRLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNRASRAELLRLEPEDPAHKPPSPHDLVYFEKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNC THTRVLHECLSeq ID No:2 Human Wnt-1 peptide sequence #2 39 NVASSTNLLTDSKS(C) 52 SeqID No:3 Human Wnt-1 peptide sequence #3 131 SAGVTHSVARSC 142 Seq ID No:4Human Wnt-1 peptide sequence #4 200 HNNEAGRTTVFS(C) 212 Seq ID No:5Human Wnt-1 peptide sequence #5 274 LEPEDPAHKPPSP(C) 286 Seq ID No:6Human Wnt-1 peptide sequence #6 332 DGCELLCCGRGHRTRTQRVTERC 347 Seq IDNo:7 Human Wnt-1 peptide sequence #7 354 HVSCRNCTHTRVLHECL 370 Seq IDNo:8 Human Wnt-2 peptide sequence #1MNAPLGGIWLWLPLLLTWLTPEVNSSWWYMRATGGSSRVMCDNVPGLVSSQRQLCHRHPDVMRAISQGVAEWTAECQHQFRQHRWNCNTLDRDHSLFGRVLLRSSRESAFVYAISSAGVVFAITRACSQGEVKSCSCDPKKMGSAKDSKGIFDWGGCSDNIDYGIKFARAFVDAKERKGKDARALMNLHNNRAGRKAVKRFLKQECKCHGVSGSCTLRTCWLAMADFRKTGDYLWRKYNGAIQVVMNQDGTGFTVANERFKKPTKNDLVYFENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKFHWCCAVRCQDCLEALDVHTCKAPKNADWTTAT Seq ID No:9Human Wnt-2 peptide sequence #2 49 SSQRQLCHRHPDVMR 63 Seq ID No:10 HumanWnt-2 peptide sequence #3 137 CDPKKMGSAKDSKG150 Seq ID No:11 Human Wnt-2peptide sequence #4 171 VDAKERKGKDAR(C) 183 Seq ID No:12 Human Wnt-2peptide sequence #5 344 DVHTCKAPKNADWTTAT(C) 360 Seq ID No:13 HumanWnt-3 peptide sequence #1MEPHLLGLLLGLLLGGTRVLAGYPIWWSLALGQQYTSLGSQPLLCGSIPGLVPKQLRFCRNYIEIMPSVAEGVKLGIQECQHQFRGRRWNCTTIDDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTSTICGCDSHHKGPPGEGWKWGGCSEDADFGVLVSREFADARENRPDARSAMNKHNNEAGRTTILDHMHLKCKCHGLSGSCEVKTCWWAQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRAKYSLFKPPTERDLVYYENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCIFHWCCYVSCQECIRIYDVHTCK Seq ID No:14Human Wnt-3A peptide sequence #1MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK Seq ID No:15 HumanWnt-4 peptide sequenceMSPRSCLRSLRLLVFAVFSAAASNWLYLAKLSSVGSISEEETCEKLKGLIQRQVQMCKRNLEVMDSVRRGAQLAIEECQYQFRNRRWNCSTLDSLPVFGKVVTQGTREAAFVYAISSAGVAFAVTRACSSGELEKCGCDRTVHGVSPQGFQWSGCSDNIAYGVAFSQSFVDVRERSKGASSSRALMNLHNNEAGRKAILTHMRVECKCHGVSGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRVGSSRALVPRNAQFKPHTDEDLVYLEPSPDFCEQDMRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCSCKFHWCCFVKCRQCQRLVELHTCR Seq ID No:16 HumanWnt-5A peptide sequenceMAGSAMSSKFFLVALAIFFSFAQVVIEANSWWSLGMNNPVQMSEVYIIGAQPLCSQLAGLSQGQKKLCHLYQDHMQYIGEGAKTGIKECQYQFRHRRWNCSTVDNTSVFGRVMQIGSRETAFTYAVSAAGVVNAMSRACREGELSTCGCSRAARPKDLPRDWLWGGCGDNIDYGYRFAKEFVDARERERIHAKGSYESARILMNLHNNEAGRRTVYNLADVACKCHGVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMRLNSRGKLVQVNSRFNSPTTQDLVYIDPSPDYCVRNESTGSLGTQGRLCNKTSEGMDGCELMCCGRGYDQFKTVQTERCHCKFHWCCYVKCKKCTEIVD QFVCK SeqID No:17 Human Wnt-5B peptide sequenceMPSLLLLFTAALLSSWAQLLTDANSWWSLALNPVQRPEMFIIGAQPVCSQLPGLSPGQRKLCQLYQEHMAYIGEGAKTGIKECQHQFRQRRWNCSTADNASVFGRVMQIGSRETAFTHAVSAAGVVNAISRACREGELSTCGCSRTARPKDLPRDWLWGGCGDNVEYGYRFAKEFVDAREREKNFAKGSEEQGRVLMNLQNNEAGRRAVYKMADVACKCHGVSGSCSLKTCWLQLAEFRKVGDRLKEKYDSAAAMRVTRKGRLELVNSRFTQPTPEDLVYVDPSPDYCLRNESTGSLGTQGRLCNKTSEGMDGCELMCCGRGYNQFKSVQVERCHCKFHWCCFVRCKKCTEIVDQYICK Seq ID No:18Human Wnt-6 peptide sequenceMLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQDIRETAFVFAITAAGASHAVTQACSMGELLQCGCQAPRGRAPPRPSGLPGTPGPPGPAGSPEGSAAWEWGGCGDDVDFGDEKSRLFMDARHKRGRGDIRALVQLHNNEAGRLAVRSHTRTECKCHGLSGSCALRTCWQKLPPFREVGARLLERFHGASRVMGTNDGKALLPAVRTLKPPGRADLLYAADSPDFCAPNRRTGSPGTRGRACNSSAPDLSGCDLLCCGRGHRQESVQLEENCLCRFHWCCVVQCHRCRVRKE LSLCL SeqID No:19 Human Wnt-7A peptide sequenceMNRKALRCLGHLFLSLGMVCLRIGGFSSVVALGATIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK Seq ID No:20 HumanWnt-7B peptide sequenceMHRNFRKWIFYVFLCFGVLYVKLGALSSVVALGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSERTEVFTCK Seq ID No:21 HumanWnt-8A peptide sequenceMGNLFMLWAALGICCAAFSASAWSVNNFLITGPKAYLTYTTSVALGAQSGIEECKFQFAWERWNCPENALQLSTHNRLRSATRETSFIHAISSAGVMYIITKNCSMGDFENCGCDGSNNGKTGGHGWIWGGCSDNVEFGERISKLFVDSLEKGKDARALMNLHNNRAGRLAVRATMKRTCKCHGISGSCSIQTCWLQLAEFREMGDYLKAKYDQALKIEMDKRQLRAGNSAEGHWVPAEAFLPSAEAELIFLEESPDYCTCNSSLGIYGTEGRECLQNSHNTSRWERRSCGRLCTECGLQVEERKTEVISSCNCKFQWCCTVKCDQCRHVVSKYYCARSPGSAQSLGRVWFGVYI Seq ID No:22Human Wnt-8B peptide sequenceMFLSKPSVYICLFTCVLQLSHSWSVNNFLMTGPKAYLIYSSSVAAGAQSGIEECKYQFAWDRWNCPERALQLSSHGGLRSANRETAFVHAISSAGVMYTLTRNCSLGDFDNCGCDDSRNGQLGGQGWLWGGCSDNVGFGEAISKQFVDALETGQDARAAMNLHNNEAGRKAVKGTMKRTCKCHGVSGSCTTQTCWLQLPEFREVGAHLKEKYHAALKVDLLQGAGNSAAARGAIADTFRSISTRELVHLEDSPDYCLENKTLGLLGTEGRECLRRGRALGRWELRSCRRLCGDCGLAVEERRAETVSSCNCKFHWCCAVRCEQCRRRVTKYFCSRAERPRGGAAHKPGRKP Seq ID No:23 HumanWnt-10A peptide sequenceMGSAHPRPWLRLRPQPQPRPALWVLLFFLLLLAAAMPRSAPNDILDLRLPPEPVLNANTVCLTLPGLSRRQMEVCVRHPDVAASAIQGIQIAIHECQHQFRDQRWNCSSLETRNKIPYESPIFSRGFRESAFAYAIAAAGVVHAVSNACALGKLKACGCDASRRGDEEAFRRKLHRLQLDALQRGKGLSHGVPEHPALPTASPGLQDSWEWGGCSPDMGFGERFSKDFLDSREPHRDIHARMRLHNNRVGRQAVMENMRRKCKCHGTSGSCQLKTCWQVTPEFRTVGALLRSRFHRATLIRPHNRNGGQLEPGPAGAPSPAPGAPGPRRRASPADLVYFEKSPDFCEREPRLDSAGTVGRLCNKSSAGSDGCGSMCCGRGHNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCK Seq ID No:24Human Wnt-10B peptide sequenceMLEEPRPRPPPSGLAGLLFLALCSRALSNEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFSFSMLAAGVMHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVTENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCK Seq ID No:25 Human Wnt-11 peptide sequenceMRARPQVCEALLFALALQTGVCYGIKWLALSKTPSALALNQTQHCKQLEGLVSAQVQLCRSNLELMHTVVHAAREVMKACRRAFADMRWNCSSIELAPNYLLDLERGTRESAFVYALSAATISHAIARACTSGDLPGCSCGPVPGEPPGPGNRWGRCADNLSYGLLMGAKFSDAPMKVKKTGSQANKLMRLHNSEVGRQALRASLEMKCKCHGVSGSCSIRTCWKGLQELQDVAADLKTRYLSATKVVHRPMGTRKHLVPKDLDIRPVKDWELVYLQSSPDFCMKNEKVGSHGTQDRQCNKTSNGSDSCDLMCCGRGYNPYTDRVVERCHCKYHWCCYVTCRRCERTVERYVCK Seq ID No:26Human Wnt-12 peptide sequenceMLEEPRPRPPPSGLAGLLFLALCSRALSNEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFSFSMLAAGVMHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVTENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCK Seq ID No:27 Human Wnt-13 peptide sequenceMLRPGGAEEAAQLPLRRASAPVPVPSPAAPDGSRASARLGLACLLLLLLLTLPARVDTSWWYIGALGARVICDNIPGLVSRQRQLCQRYPDIMRSVGEGAREWIRECQHQFRHHRWNCTTLDRDHTVFGRVMLRSSREAAFVYAISSAGVVHAITRACSQGELSVCSCDPYTRGRHHDQRGDFDWGGCSDNIHYGVRFAKAFVDAKEKRLKDARALMNLHNNRCGRTAVRRFLKLECKCHGVSGSCTLRTCWRALSDFRRTGDYLRRRYDGAVQVMATQDGANFTAARQGYRRATRTDLVYFDNSPDYCVLDKAAGSLGTAGRVCSKTSKGTDGCEIMCCGRGYDTTRVTRVTQCECKFHWCCAVRCKECRNTVDVHTCKAPKKAEWLDQT Seq ID No:28 Human Wnt-14 peptidesequence MLDGSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEV YTCKG SeqID No:29 Human Wnt-15 peptide sequenceMRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH Seq ID No:30Human Wnt-16 peptide sequenceMERHPPMQLTTCLRETLFTGASQKTSLWWLGIASFGVPEKLGCANLPLNSRQKELCKRKPYLLPSIREGARLGIQECRSQFRHERWNCMITAAATTAPMGASPLFGYELSSGTKETAFIYAVMAAGLVHSVTRSCSAGNMTECSCDTTLQNGGSASEGWHWGGCSDDVQYGMWFSRKFLDFPIGNTTGKENKVLLAMNLHNNEAGRQAVAKLMSVDCRCHGVSGSCAVKTCWKTMSSFEKIGHLLKDKYENSIQISDKIKRKMRRREKDQRKIPIHKDDLLYVNKSPNYCVEDKKLGIPGTQGRECNRTSEGADGCNLLCCGRGYNTHVVRHVERCECKFIWCCYVRCRRCESMTDVHTCK Seq IDNo:31 Human Frizzled-1 peptide sequence (extracellularcysteine-rich domain)MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWLLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYGLMYFGPEELR Seq ID No:32 Human Frizzled-2 peptide sequence(extracellular cysteine-rich domain)MRPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEE TR Seq IDNo:33 Human Frizzled-3 peptide sequenceMAMTWIVFSLWPLTVFMGHIGGHSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPYPRLVDLNLAGEPTEGAPVAVQRDYGFWCPRELKIDPDLGYSFLHVRDCSPPCPNMYFRREELS Seq ID No:34 Human Frizzled-4 peptide sequenceMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKE Seq ID No:35 Human Frizzled-5peptide sequenceMARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCTMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPFVPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERT Seq ID No:36 HumanFrizzled-6 peptide sequenceMEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGPQKKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELE Seq ID No:37 Human Frizzled-7 peptide sequenceMRDPGAAVPLSSLGFCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGKGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRAN GLMYFKEEERRSeq ID No:38 Human Frizzled-8 peptide sequenceMEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPRRLPPPPPGEQPPSGSGHGRPPGARPPHRGGGRGGGGGDAAAPPARGGGGGGKARPPGGGAAPCEPGCQCRAPMVSVSSERHPLYNRVKTGQIANCALPCHNPFFSQDERA Seq ID No:39 Human Frizzled-9peptide sequenceMAVAPLRGALLLWQLLAAGGAALEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENATAGPAEPHKGLGMLPVAPRPARPPGDLGPGAGGSGTCENPEKFQYVEKSRSCAPRCGPGVEVFWSRRDKD Seq ID No:40 HumanFrizzled-10 peptide sequenceMQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHHVEKSASCAPLCTPGVDVYWSREDKR SEQ ID NO:41 Human DVL-3amino acid sequence MGETKIIYHL DGQETPYLVK LPLPAERVTL ADFKGVLQRPSYKFFFKSMD DDFGVVKEEI SDDNAKLPCF NGRVVYWLVS AEGSHPDPAP FCADNPSELPPPMERTGGIG DSRPPSFHPH AGGGSQENLD NDTETDSLVS AQRERPRRRD GPEHATRLNGTAKGERRREP GGYDSSSTLM SSELETTSFF DSDEDDSTSR FSSSTEQSSA SRLMRRHKRRRRKQKVSRIE RSSSFSSITD STMSLNIITV TLNMEKYNFL GISIVGQSNE RGDGGIYIGSIMKGGAVAAD GRIEPGDMLL QVNEINFENM SNDDAVRVLR EIVHKPGPIT LTVAKCWDPSPRGCFTLPRS EPIRPIDPAA WVSHTAAMTG TFPAYGMSPS LSTITSTSSS ITSSIPDTERLDDFHLSIHS DMAAIVKAMA SPESGLEVRD RMWLKITIPN AFIGSDVVDW LYHNVEGFTDRREARKYASN LLKAGFIRHT VNKITFSEQG YYIFGDLCGN MANLSLHDHD GSSGASDQDTLAPLPHPGAA PWPMAFPYQY PPPPHPYNPH PGFPELGYSY GGGSASSQHS EGSRSSGSNRSGSDRRKEKD PKAGDSKSGG SGSESDHTTR SSLRGPRERA PSERSGPAAS EHSHRSHHSLASSLRSHHTH PSYGPPGVPP LYGPPMLMMP PPPAAMGPPG APPGRDLASV PPELTASRQSFRMAMGNPSE FFVDVM SEQ ID NO:42: Human Dvl-1 amino acid sequenceMAETKIIYHM DEEETPYLVK LPVAPERVTL ADFKNVLSNR PVHAYKFFFK SMDQDFGVVKEEIFDDNAKL PCFNGRVVSW LVLAEGAHSD AGSQGTDSHT DLPPPLERTG GIGDSRPPSFHPNVASSRDG MDNETGTESM VSHRRERARR RNREEAARTN GHPRGDRRRD VGLPPDSASTALSSELESSS FVDSDEDGST SRLSSSTEQS TSSRLIRKHK RRRRKQRLRQ ADRASSFSSITDSTMSLNIV TVTLNMERHH FLGISIVGQS NDRGDGGIYI GSIMKGGAVA ADGRIEPGDMLLQVNDVNFE NMSNDDAVRV LREIVSQTGP ISLTVAKCWD PTPRSYFTVP RADPVRPIDPAAWLSHTAAL TGALPRYELE EAPLTVKSDM SAVVRVMQLP DSGLEIRDRM WLKITIANAVIGADVVDWLY THVEGFKERR EARKYASSLL KHGFLRHTVN KITFSEQCYY VFGDLCSNLATLNLNSGSSG TSDQDTLAPL PHPAAPWPLG QGYPYQYPGP PPCFPPAYQD PGFSYGSGSTGSQQSEGSKS SGSTRSSRRA PGREKERRAA GAGGSGSESD HTAPSGVGSS WRERPAGQLSRGSSPRSQAS ATAPGLPPPH PTTKAYTVVG GPPGGPPVRE LAAVPPELTG SRQSFQKAMGNPCEFFVDIM SEQ ID NO:43: Human Dvl-2 amino acid sequence MAGSSTGGGGVGETKVIYHL DEEETPYLVK IPVPAERITL GDFKSVLQRP AGAKYFFKSM DQDFGVVKEEISDDNARLPC FNGRVVSWLV SSDNPQPEMA PPVHEPRAEL APPAPPLPPL PPERTSGIGDSRPPSFHPNV SSSHENLEPE TETESVVSLR RERPRRRDSS EHGAGGHRTG GPSRLERHLAGYESSSTLMT SELESTSLGD SDEEDTMSRF SSSTEQSSAS RLLKRHRRRR KQRPPRLERTSSFSSVTDST MSLNIITVTL NMEKYNFLGI SIVGQSNERG DGGIYIGSIM KGGAVAADGRIEPGDMLLQV NDMNFENMSN DDAVRVLRDI VHKPGPIVLT VAKCWDPSPQ AYFTLPRNEPIQPIDPAAWV SHSAALTGTF PAYPGSSSMS TITSGSSLPD GCEGRGLSVH TDMASVTKAMAAPESGLEVR DRMWLKITIP NAFLGSDVVD WLYHHVEGFP ERREARKYAS GLLKAGLIRHTVNKITFSEQ CYYVFGDLSG GCESYLVNLS LNDNDGSSGA SDQDTLAPLP GATPWPLLPTFSYQYPAPHP YSPQPPPYHE LSSYTYGGGS ASSQHSEGSR SSGSTRSDGG AGRTGRPEERAPESKSGSGS ESEPSSRGGS LRRGGEASGT SDGGPPPSRG STGGAPNLRA HPGLHPYGPPPGMALPYNPM MVVMMPPPPP PVPPAVQPPG APPVRDLGSV PPELTASRQS FHMAMGNPSE FFVDVM

[0213]

1 80 1 370 PRT Homo sapiens human Wingless-type 1 (Wnt-1) peptidesequence #1 1 Met Gly Leu Trp Ala Leu Leu Pro Gly Trp Val Ser Ala ThrLeu Leu 1 5 10 15 Leu Ala Leu Ala Ala Leu Pro Ala Ala Leu Ala Ala AsnSer Ser Gly 20 25 30 Arg Trp Trp Gly Ile Val Asn Val Ala Ser Ser Thr AsnLeu Leu Thr 35 40 45 Asp Ser Lys Ser Leu Gln Leu Val Leu Glu Pro Ser LeuGln Leu Leu 50 55 60 Ser Arg Lys Gln Arg Arg Leu Ile Arg Gln Asn Pro GlyIle Leu His 65 70 75 80 Ser Val Ser Gly Gly Leu Gln Ser Ala Val Arg GluCys Lys Trp Gln 85 90 95 Phe Arg Asn Arg Arg Trp Asn Cys Pro Thr Ala ProGly Pro His Leu 100 105 110 Phe Gly Lys Ile Val Asn Arg Gly Cys Arg GluThr Ala Phe Ile Phe 115 120 125 Ala Ile Thr Ser Ala Gly Val Thr His SerVal Ala Arg Ser Cys Ser 130 135 140 Glu Gly Ser Ile Glu Ser Cys Thr CysAsp Tyr Arg Arg Arg Gly Pro 145 150 155 160 Gly Gly Pro Asp Trp His TrpGly Gly Cys Ser Asp Asn Ile Asp Phe 165 170 175 Gly Arg Leu Phe Gly ArgGlu Phe Val Asp Ser Gly Glu Lys Gly Arg 180 185 190 Asp Leu Arg Phe LeuMet Asn Leu His Asn Asn Glu Ala Gly Arg Thr 195 200 205 Thr Val Phe SerGlu Met Arg Gln Glu Cys Lys Cys His Gly Met Ser 210 215 220 Gly Ser CysThr Val Arg Thr Cys Trp Met Arg Leu Pro Thr Leu Arg 225 230 235 240 AlaVal Gly Asp Val Leu Arg Asp Arg Phe Asp Gly Ala Ser Arg Val 245 250 255Leu Tyr Gly Asn Arg Gly Ser Asn Arg Ala Ser Arg Ala Glu Leu Leu 260 265270 Arg Leu Glu Pro Glu Asp Pro Ala His Lys Pro Pro Ser Pro His Asp 275280 285 Leu Val Tyr Phe Glu Lys Ser Pro Asn Phe Cys Thr Tyr Ser Gly Arg290 295 300 Leu Gly Thr Ala Gly Thr Ala Gly Arg Ala Cys Asn Ser Ser SerPro 305 310 315 320 Ala Leu Asp Gly Cys Glu Leu Leu Cys Cys Gly Arg GlyHis Arg Thr 325 330 335 Arg Thr Gln Arg Val Thr Glu Arg Cys Asn Cys ThrPhe His Trp Cys 340 345 350 Cys His Val Ser Cys Arg Asn Cys Thr His ThrArg Val Leu His Glu 355 360 365 Cys Leu 370 2 15 PRT Homo sapiens humanWingless-type 1 (Wnt-1) peptide sequence #2 2 Asn Val Ala Ser Ser ThrAsn Leu Leu Thr Asp Ser Lys Ser Cys 1 5 10 15 3 12 PRT Homo sapienshuman Wingless-type 1 (Wnt-1) peptide sequence #3 3 Ser Ala Gly Val ThrHis Ser Val Ala Arg Ser Cys 1 5 10 4 13 PRT Homo sapiens humanWingless-type 1 (Wnt-1) peptide sequence #4 4 His Asn Asn Glu Ala GlyArg Thr Thr Val Phe Ser Cys 1 5 10 5 14 PRT Homo sapiens humanWingless-type 1 (Wnt-1) peptide sequence #5 5 Leu Glu Pro Glu Asp ProAla His Lys Pro Pro Ser Pro Cys 1 5 10 6 23 PRT Homo sapiens humanWingless-type 1 (Wnt-1) peptide sequence #6 6 Asp Gly Cys Glu Leu LeuCys Cys Gly Arg Gly His Arg Thr Arg Thr 1 5 10 15 Gln Arg Val Thr GluArg Cys 20 7 17 PRT Homo sapiens human Wingless-type 1 (Wnt-1) peptidesequence #7 7 His Val Ser Cys Arg Asn Cys Thr His Thr Arg Val Leu HisGlu Cys 1 5 10 15 Leu 8 360 PRT Homo sapiens human Wingless-type 2(Wnt-2) peptide sequence #1 8 Met Asn Ala Pro Leu Gly Gly Ile Trp LeuTrp Leu Pro Leu Leu Leu 1 5 10 15 Thr Trp Leu Thr Pro Glu Val Asn SerSer Trp Trp Tyr Met Arg Ala 20 25 30 Thr Gly Gly Ser Ser Arg Val Met CysAsp Asn Val Pro Gly Leu Val 35 40 45 Ser Ser Gln Arg Gln Leu Cys His ArgHis Pro Asp Val Met Arg Ala 50 55 60 Ile Ser Gln Gly Val Ala Glu Trp ThrAla Glu Cys Gln His Gln Phe 65 70 75 80 Arg Gln His Arg Trp Asn Cys AsnThr Leu Asp Arg Asp His Ser Leu 85 90 95 Phe Gly Arg Val Leu Leu Arg SerSer Arg Glu Ser Ala Phe Val Tyr 100 105 110 Ala Ile Ser Ser Ala Gly ValVal Phe Ala Ile Thr Arg Ala Cys Ser 115 120 125 Gln Gly Glu Val Lys SerCys Ser Cys Asp Pro Lys Lys Met Gly Ser 130 135 140 Ala Lys Asp Ser LysGly Ile Phe Asp Trp Gly Gly Cys Ser Asp Asn 145 150 155 160 Ile Asp TyrGly Ile Lys Phe Ala Arg Ala Phe Val Asp Ala Lys Glu 165 170 175 Arg LysGly Lys Asp Ala Arg Ala Leu Met Asn Leu His Asn Asn Arg 180 185 190 AlaGly Arg Lys Ala Val Lys Arg Phe Leu Lys Gln Glu Cys Lys Cys 195 200 205His Gly Val Ser Gly Ser Cys Thr Leu Arg Thr Cys Trp Leu Ala Met 210 215220 Ala Asp Phe Arg Lys Thr Gly Asp Tyr Leu Trp Arg Lys Tyr Asn Gly 225230 235 240 Ala Ile Gln Val Val Met Asn Gln Asp Gly Thr Gly Phe Thr ValAla 245 250 255 Asn Glu Arg Phe Lys Lys Pro Thr Lys Asn Asp Leu Val TyrPhe Glu 260 265 270 Asn Ser Pro Asp Tyr Cys Ile Arg Asp Arg Glu Ala GlySer Leu Gly 275 280 285 Thr Ala Gly Arg Val Cys Asn Leu Thr Ser Arg GlyMet Asp Ser Cys 290 295 300 Glu Val Met Cys Cys Gly Arg Gly Tyr Asp ThrSer His Val Thr Arg 305 310 315 320 Met Thr Lys Cys Gly Cys Lys Phe HisTrp Cys Cys Ala Val Arg Cys 325 330 335 Gln Asp Cys Leu Glu Ala Leu AspVal His Thr Cys Lys Ala Pro Lys 340 345 350 Asn Ala Asp Trp Thr Thr AlaThr 355 360 9 15 PRT Homo sapiens human Wingless-type 2 (Wnt-2) peptidesequence #2, amino acids 49-63 of human Wnt-2 9 Ser Ser Gln Arg Gln LeuCys His Arg His Pro Asp Val Met Arg 1 5 10 15 10 14 PRT Homo sapienshuman Wingless-type 2 (Wnt-2) peptide sequence #3 10 Cys Asp Pro Lys LysMet Gly Ser Ala Lys Asp Ser Lys Gly 1 5 10 11 13 PRT Homo sapiens humanWingless-type 2 (Wnt-2) peptide sequence #4 11 Val Asp Ala Lys Glu ArgLys Gly Lys Asp Ala Arg Cys 1 5 10 12 18 PRT Homo sapiens humanWingless-type 2 (Wnt-2) peptide sequence #5 12 Asp Val His Thr Cys LysAla Pro Lys Asn Ala Asp Trp Thr Thr Ala 1 5 10 15 Thr Cys 13 355 PRTHomo sapiens human Wingless-type 3 (Wnt-3) peptide sequence #1 13 MetGlu Pro His Leu Leu Gly Leu Leu Leu Gly Leu Leu Leu Gly Gly 1 5 10 15Thr Arg Val Leu Ala Gly Tyr Pro Ile Trp Trp Ser Leu Ala Leu Gly 20 25 30Gln Gln Tyr Thr Ser Leu Gly Ser Gln Pro Leu Leu Cys Gly Ser Ile 35 40 45Pro Gly Leu Val Pro Lys Gln Leu Arg Phe Cys Arg Asn Tyr Ile Glu 50 55 60Ile Met Pro Ser Val Ala Glu Gly Val Lys Leu Gly Ile Gln Glu Cys 65 70 7580 Gln His Gln Phe Arg Gly Arg Arg Trp Asn Cys Thr Thr Ile Asp Asp 85 9095 Ser Leu Ala Ile Phe Gly Pro Val Leu Asp Lys Ala Thr Arg Glu Ser 100105 110 Ala Phe Val His Ala Ile Ala Ser Ala Gly Val Ala Phe Ala Val Thr115 120 125 Arg Ser Cys Ala Glu Gly Thr Ser Thr Ile Cys Gly Cys Asp SerHis 130 135 140 His Lys Gly Pro Pro Gly Glu Gly Trp Lys Trp Gly Gly CysSer Glu 145 150 155 160 Asp Ala Asp Phe Gly Val Leu Val Ser Arg Glu PheAla Asp Ala Arg 165 170 175 Glu Asn Arg Pro Asp Ala Arg Ser Ala Met AsnLys His Asn Asn Glu 180 185 190 Ala Gly Arg Thr Thr Ile Leu Asp His MetHis Leu Lys Cys Lys Cys 195 200 205 His Gly Leu Ser Gly Ser Cys Glu ValLys Thr Cys Trp Trp Ala Gln 210 215 220 Pro Asp Phe Arg Ala Ile Gly AspPhe Leu Lys Asp Lys Tyr Asp Ser 225 230 235 240 Ala Ser Glu Met Val ValGlu Lys His Arg Glu Ser Arg Gly Trp Val 245 250 255 Glu Thr Leu Arg AlaLys Tyr Ser Leu Phe Lys Pro Pro Thr Glu Arg 260 265 270 Asp Leu Val TyrTyr Glu Asn Ser Pro Asn Phe Cys Glu Pro Asn Pro 275 280 285 Glu Thr GlySer Phe Gly Thr Arg Asp Arg Thr Cys Asn Val Thr Ser 290 295 300 His GlyIle Asp Gly Cys Asp Leu Leu Cys Cys Gly Arg Gly His Asn 305 310 315 320Thr Arg Thr Glu Lys Arg Lys Glu Lys Cys His Cys Ile Phe His Trp 325 330335 Cys Cys Tyr Val Ser Cys Gln Glu Cys Ile Arg Ile Tyr Asp Val His 340345 350 Thr Cys Lys 355 14 352 PRT Homo sapiens human Wingless-type 3A(Wnt-3A) peptide sequence #1 14 Met Ala Pro Leu Gly Tyr Phe Leu Leu LeuCys Ser Leu Lys Gln Ala 1 5 10 15 Leu Gly Ser Tyr Pro Ile Trp Trp SerLeu Ala Val Gly Pro Gln Tyr 20 25 30 Ser Ser Leu Gly Ser Gln Pro Ile LeuCys Ala Ser Ile Pro Gly Leu 35 40 45 Val Pro Lys Gln Leu Arg Phe Cys ArgAsn Tyr Val Glu Ile Met Pro 50 55 60 Ser Val Ala Glu Gly Ile Lys Ile GlyIle Gln Glu Cys Gln His Gln 65 70 75 80 Phe Arg Gly Arg Arg Trp Asn CysThr Thr Val His Asp Ser Leu Ala 85 90 95 Ile Phe Gly Pro Val Leu Asp LysAla Thr Arg Glu Ser Ala Phe Val 100 105 110 His Ala Ile Ala Ser Ala GlyVal Ala Phe Ala Val Thr Arg Ser Cys 115 120 125 Ala Glu Gly Thr Ala AlaIle Cys Gly Cys Ser Ser Arg His Gln Gly 130 135 140 Ser Pro Gly Lys GlyTrp Lys Trp Gly Gly Cys Ser Glu Asp Ile Glu 145 150 155 160 Phe Gly GlyMet Val Ser Arg Glu Phe Ala Asp Ala Arg Glu Asn Arg 165 170 175 Pro AspAla Arg Ser Ala Met Asn Arg His Asn Asn Glu Ala Gly Arg 180 185 190 GlnAla Ile Ala Ser His Met His Leu Lys Cys Lys Cys His Gly Leu 195 200 205Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Trp Ser Gln Pro Asp Phe 210 215220 Arg Ala Ile Gly Asp Phe Leu Lys Asp Lys Tyr Asp Ser Ala Ser Glu 225230 235 240 Met Val Val Glu Lys His Arg Glu Ser Arg Gly Trp Val Glu ThrLeu 245 250 255 Arg Pro Arg Tyr Thr Tyr Phe Lys Val Pro Thr Glu Arg AspLeu Val 260 265 270 Tyr Tyr Glu Ala Ser Pro Asn Phe Cys Glu Pro Asn ProGlu Thr Gly 275 280 285 Ser Phe Gly Thr Arg Asp Arg Thr Cys Asn Val SerSer His Gly Ile 290 295 300 Asp Gly Cys Asp Leu Leu Cys Cys Gly Arg GlyHis Asn Ala Arg Ala 305 310 315 320 Glu Arg Arg Arg Glu Lys Cys Arg CysVal Phe His Trp Cys Cys Tyr 325 330 335 Val Ser Cys Gln Glu Cys Thr ArgVal Tyr Asp Val His Thr Cys Lys 340 345 350 15 351 PRT Homo sapienshuman Wingless-type 4 (Wnt-4) peptide sequence 15 Met Ser Pro Arg SerCys Leu Arg Ser Leu Arg Leu Leu Val Phe Ala 1 5 10 15 Val Phe Ser AlaAla Ala Ser Asn Trp Leu Tyr Leu Ala Lys Leu Ser 20 25 30 Ser Val Gly SerIle Ser Glu Glu Glu Thr Cys Glu Lys Leu Lys Gly 35 40 45 Leu Ile Gln ArgGln Val Gln Met Cys Lys Arg Asn Leu Glu Val Met 50 55 60 Asp Ser Val ArgArg Gly Ala Gln Leu Ala Ile Glu Glu Cys Gln Tyr 65 70 75 80 Gln Phe ArgAsn Arg Arg Trp Asn Cys Ser Thr Leu Asp Ser Leu Pro 85 90 95 Val Phe GlyLys Val Val Thr Gln Gly Thr Arg Glu Ala Ala Phe Val 100 105 110 Tyr AlaIle Ser Ser Ala Gly Val Ala Phe Ala Val Thr Arg Ala Cys 115 120 125 SerSer Gly Glu Leu Glu Lys Cys Gly Cys Asp Arg Thr Val His Gly 130 135 140Val Ser Pro Gln Gly Phe Gln Trp Ser Gly Cys Ser Asp Asn Ile Ala 145 150155 160 Tyr Gly Val Ala Phe Ser Gln Ser Phe Val Asp Val Arg Glu Arg Ser165 170 175 Lys Gly Ala Ser Ser Ser Arg Ala Leu Met Asn Leu His Asn AsnGlu 180 185 190 Ala Gly Arg Lys Ala Ile Leu Thr His Met Arg Val Glu CysLys Cys 195 200 205 His Gly Val Ser Gly Ser Cys Glu Val Lys Thr Cys TrpArg Ala Val 210 215 220 Pro Pro Phe Arg Gln Val Gly His Ala Leu Lys GluLys Phe Asp Gly 225 230 235 240 Ala Thr Glu Val Glu Pro Arg Arg Val GlySer Ser Arg Ala Leu Val 245 250 255 Pro Arg Asn Ala Gln Phe Lys Pro HisThr Asp Glu Asp Leu Val Tyr 260 265 270 Leu Glu Pro Ser Pro Asp Phe CysGlu Gln Asp Met Arg Ser Gly Val 275 280 285 Leu Gly Thr Arg Gly Arg ThrCys Asn Lys Thr Ser Lys Ala Ile Asp 290 295 300 Gly Cys Glu Leu Leu CysCys Gly Arg Gly Phe His Thr Ala Gln Val 305 310 315 320 Glu Leu Ala GluArg Cys Ser Cys Lys Phe His Trp Cys Cys Phe Val 325 330 335 Lys Cys ArgGln Cys Gln Arg Leu Val Glu Leu His Thr Cys Arg 340 345 350 16 365 PRTHomo sapiens human Wingless-type 5A (Wnt-5A) peptide sequence 16 Met AlaGly Ser Ala Met Ser Ser Lys Phe Phe Leu Val Ala Leu Ala 1 5 10 15 IlePhe Phe Ser Phe Ala Gln Val Val Ile Glu Ala Asn Ser Trp Trp 20 25 30 SerLeu Gly Met Asn Asn Pro Val Gln Met Ser Glu Val Tyr Ile Ile 35 40 45 GlyAla Gln Pro Leu Cys Ser Gln Leu Ala Gly Leu Ser Gln Gly Gln 50 55 60 LysLys Leu Cys His Leu Tyr Gln Asp His Met Gln Tyr Ile Gly Glu 65 70 75 80Gly Ala Lys Thr Gly Ile Lys Glu Cys Gln Tyr Gln Phe Arg His Arg 85 90 95Arg Trp Asn Cys Ser Thr Val Asp Asn Thr Ser Val Phe Gly Arg Val 100 105110 Met Gln Ile Gly Ser Arg Glu Thr Ala Phe Thr Tyr Ala Val Ser Ala 115120 125 Ala Gly Val Val Asn Ala Met Ser Arg Ala Cys Arg Glu Gly Glu Leu130 135 140 Ser Thr Cys Gly Cys Ser Arg Ala Ala Arg Pro Lys Asp Leu ProArg 145 150 155 160 Asp Trp Leu Trp Gly Gly Cys Gly Asp Asn Ile Asp TyrGly Tyr Arg 165 170 175 Phe Ala Lys Glu Phe Val Asp Ala Arg Glu Arg GluArg Ile His Ala 180 185 190 Lys Gly Ser Tyr Glu Ser Ala Arg Ile Leu MetAsn Leu His Asn Asn 195 200 205 Glu Ala Gly Arg Arg Thr Val Tyr Asn LeuAla Asp Val Ala Cys Lys 210 215 220 Cys His Gly Val Ser Gly Ser Cys SerLeu Lys Thr Cys Trp Leu Gln 225 230 235 240 Leu Ala Asp Phe Arg Lys ValGly Asp Ala Leu Lys Glu Lys Tyr Asp 245 250 255 Ser Ala Ala Ala Met ArgLeu Asn Ser Arg Gly Lys Leu Val Gln Val 260 265 270 Asn Ser Arg Phe AsnSer Pro Thr Thr Gln Asp Leu Val Tyr Ile Asp 275 280 285 Pro Ser Pro AspTyr Cys Val Arg Asn Glu Ser Thr Gly Ser Leu Gly 290 295 300 Thr Gln GlyArg Leu Cys Asn Lys Thr Ser Glu Gly Met Asp Gly Cys 305 310 315 320 GluLeu Met Cys Cys Gly Arg Gly Tyr Asp Gln Phe Lys Thr Val Gln 325 330 335Thr Glu Arg Cys His Cys Lys Phe His Trp Cys Cys Tyr Val Lys Cys 340 345350 Lys Lys Cys Thr Glu Ile Val Asp Gln Phe Val Cys Lys 355 360 365 17359 PRT Homo sapiens human Wingless-type 5B (Wnt-5B) peptide sequence 17Met Pro Ser Leu Leu Leu Leu Phe Thr Ala Ala Leu Leu Ser Ser Trp 1 5 1015 Ala Gln Leu Leu Thr Asp Ala Asn Ser Trp Trp Ser Leu Ala Leu Asn 20 2530 Pro Val Gln Arg Pro Glu Met Phe Ile Ile Gly Ala Gln Pro Val Cys 35 4045 Ser Gln Leu Pro Gly Leu Ser Pro Gly Gln Arg Lys Leu Cys Gln Leu 50 5560 Tyr Gln Glu His Met Ala Tyr Ile Gly Glu Gly Ala Lys Thr Gly Ile 65 7075 80 Lys Glu Cys Gln His Gln Phe Arg Gln Arg Arg Trp Asn Cys Ser Thr 8590 95 Ala Asp Asn Ala Ser Val Phe Gly Arg Val Met Gln Ile Gly Ser Arg100 105 110 Glu Thr Ala Phe Thr His Ala Val Ser Ala Ala Gly Val Val AsnAla 115 120 125 Ile Ser Arg Ala Cys Arg Glu Gly Glu Leu Ser Thr Cys GlyCys Ser 130 135 140 Arg Thr Ala Arg Pro Lys Asp Leu Pro Arg Asp Trp LeuTrp Gly Gly 145 150 155 160 Cys Gly Asp Asn Val Glu Tyr Gly Tyr Arg PheAla Lys Glu Phe Val 165 170 175 Asp Ala Arg Glu Arg Glu Lys Asn Phe AlaLys Gly Ser Glu Glu Gln 180 185 190 Gly Arg Val Leu Met Asn Leu Gln AsnAsn Glu Ala Gly Arg Arg Ala 195 200 205 Val Tyr Lys Met Ala Asp Val AlaCys Lys Cys His Gly Val Ser Gly 210 215 220 Ser Cys Ser Leu Lys Thr CysTrp Leu Gln Leu Ala Glu Phe Arg Lys 225 230 235 240 Val Gly Asp Arg LeuLys Glu Lys Tyr Asp Ser Ala Ala Ala Met Arg 245 250 255 Val Thr Arg LysGly Arg Leu Glu Leu Val Asn Ser Arg Phe Thr Gln 260 265 270 Pro Thr ProGlu Asp Leu Val Tyr Val Asp Pro Ser Pro Asp Tyr Cys 275 280 285 Leu ArgAsn Glu Ser Thr Gly Ser Leu Gly Thr Gln Gly Arg Leu Cys 290 295 300 AsnLys Thr Ser Glu Gly Met Asp Gly Cys Glu Leu Met Cys Cys Gly 305 310 315320 Arg Gly Tyr Asn Gln Phe Lys Ser Val Gln Val Glu Arg Cys His Cys 325330 335 Lys Phe His Trp Cys Cys Phe Val Arg Cys Lys Lys Cys Thr Glu Ile340 345 350 Val Asp Gln Tyr Ile Cys Lys 355 18 365 PRT Homo sapienshuman Wingless-type 6 (Wnt-6) peptide sequence 18 Met Leu Pro Pro LeuPro Ser Arg Leu Gly Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Cys Pro AlaHis Val Gly Gly Leu Trp Trp Ala Val Gly Ser Pro 20 25 30 Leu Val Met AspPro Thr Ser Ile Cys Arg Lys Ala Arg Arg Leu Ala 35 40 45 Gly Arg Gln AlaGlu Leu Cys Gln Ala Glu Pro Glu Val Val Ala Glu 50 55 60 Leu Ala Arg GlyAla Arg Leu Gly Val Arg Glu Cys Gln Phe Gln Phe 65 70 75 80 Arg Phe ArgArg Trp Asn Cys Ser Ser His Ser Lys Ala Phe Gly Arg 85 90 95 Ile Leu GlnGln Asp Ile Arg Glu Thr Ala Phe Val Phe Ala Ile Thr 100 105 110 Ala AlaGly Ala Ser His Ala Val Thr Gln Ala Cys Ser Met Gly Glu 115 120 125 LeuLeu Gln Cys Gly Cys Gln Ala Pro Arg Gly Arg Ala Pro Pro Arg 130 135 140Pro Ser Gly Leu Pro Gly Thr Pro Gly Pro Pro Gly Pro Ala Gly Ser 145 150155 160 Pro Glu Gly Ser Ala Ala Trp Glu Trp Gly Gly Cys Gly Asp Asp Val165 170 175 Asp Phe Gly Asp Glu Lys Ser Arg Leu Phe Met Asp Ala Arg HisLys 180 185 190 Arg Gly Arg Gly Asp Ile Arg Ala Leu Val Gln Leu His AsnAsn Glu 195 200 205 Ala Gly Arg Leu Ala Val Arg Ser His Thr Arg Thr GluCys Lys Cys 210 215 220 His Gly Leu Ser Gly Ser Cys Ala Leu Arg Thr CysTrp Gln Lys Leu 225 230 235 240 Pro Pro Phe Arg Glu Val Gly Ala Arg LeuLeu Glu Arg Phe His Gly 245 250 255 Ala Ser Arg Val Met Gly Thr Asn AspGly Lys Ala Leu Leu Pro Ala 260 265 270 Val Arg Thr Leu Lys Pro Pro GlyArg Ala Asp Leu Leu Tyr Ala Ala 275 280 285 Asp Ser Pro Asp Phe Cys AlaPro Asn Arg Arg Thr Gly Ser Pro Gly 290 295 300 Thr Arg Gly Arg Ala CysAsn Ser Ser Ala Pro Asp Leu Ser Gly Cys 305 310 315 320 Asp Leu Leu CysCys Gly Arg Gly His Arg Gln Glu Ser Val Gln Leu 325 330 335 Glu Glu AsnCys Leu Cys Arg Phe His Trp Cys Cys Val Val Gln Cys 340 345 350 His ArgCys Arg Val Arg Lys Glu Leu Ser Leu Cys Leu 355 360 365 19 349 PRT Homosapiens human Wingless-type 7A (Wnt-7A) peptide sequence 19 Met Asn ArgLys Ala Leu Arg Cys Leu Gly His Leu Phe Leu Ser Leu 1 5 10 15 Gly MetVal Cys Leu Arg Ile Gly Gly Phe Ser Ser Val Val Ala Leu 20 25 30 Gly AlaThr Ile Ile Cys Asn Lys Ile Pro Gly Leu Ala Pro Arg Gln 35 40 45 Arg AlaIle Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile Gly Glu 50 55 60 Gly SerGln Met Gly Leu Asp Glu Cys Gln Phe Gln Phe Arg Asn Gly 65 70 75 80 ArgTrp Asn Cys Ser Ala Leu Gly Glu Arg Thr Val Phe Gly Lys Glu 85 90 95 LeuLys Val Gly Ser Arg Asp Gly Ala Phe Thr Tyr Ala Ile Ile Ala 100 105 110Ala Gly Val Ala His Ala Ile Thr Ala Ala Cys Thr His Gly Asn Leu 115 120125 Ser Asp Cys Gly Cys Asp Lys Glu Lys Gln Gly Gln Tyr His Arg Asp 130135 140 Glu Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Ile Arg Tyr Gly Ile145 150 155 160 Gly Phe Ala Lys Val Phe Val Asp Ala Arg Glu Ile Lys GlnAsn Ala 165 170 175 Arg Thr Leu Met Asn Leu His Asn Asn Glu Ala Gly ArgLys Ile Leu 180 185 190 Glu Glu Asn Met Lys Leu Glu Cys Lys Cys His GlyVal Ser Gly Ser 195 200 205 Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu ProGln Phe Arg Glu Leu 210 215 220 Gly Tyr Val Leu Lys Asp Lys Tyr Asn GluAla Val His Val Glu Pro 225 230 235 240 Val Arg Ala Ser Arg Asn Lys ArgPro Thr Phe Leu Lys Ile Lys Lys 245 250 255 Pro Leu Ser Tyr Arg Lys ProMet Asp Thr Asp Leu Val Tyr Ile Glu 260 265 270 Lys Ser Pro Asn Tyr CysGlu Glu Asp Pro Val Thr Gly Ser Val Gly 275 280 285 Thr Gln Gly Arg AlaCys Asn Lys Thr Ala Pro Gln Ala Ser Gly Cys 290 295 300 Asp Leu Met CysCys Gly Arg Gly Tyr Asn Thr His Gln Tyr Ala Arg 305 310 315 320 Val TrpGln Cys Asn Cys Lys Phe His Trp Cys Cys Tyr Val Lys Cys 325 330 335 AsnThr Cys Ser Glu Arg Thr Glu Met Tyr Thr Cys Lys 340 345 20 349 PRT Homosapiens human Wingless-type 7B (Wnt-7B) peptide sequence 20 Met His ArgAsn Phe Arg Lys Trp Ile Phe Tyr Val Phe Leu Cys Phe 1 5 10 15 Gly ValLeu Tyr Val Lys Leu Gly Ala Leu Ser Ser Val Val Ala Leu 20 25 30 Gly AlaAsn Ile Ile Cys Asn Lys Ile Pro Gly Leu Ala Pro Arg Gln 35 40 45 Arg AlaIle Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile Gly Glu 50 55 60 Gly AlaGln Met Gly Ile Asn Glu Cys Gln Tyr Gln Phe Arg Phe Gly 65 70 75 80 ArgTrp Asn Cys Ser Ala Leu Gly Glu Lys Thr Val Phe Gly Gln Glu 85 90 95 LeuArg Val Gly Ser Arg Glu Ala Ala Phe Thr Tyr Ala Ile Thr Ala 100 105 110Ala Gly Val Ala His Ala Val Thr Ala Ala Cys Ser Gln Gly Asn Leu 115 120125 Ser Asn Cys Gly Cys Asp Arg Glu Lys Gln Gly Tyr Tyr Asn Gln Ala 130135 140 Glu Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Val Arg Tyr Gly Ile145 150 155 160 Asp Phe Ser Arg Arg Phe Val Asp Ala Arg Glu Ile Lys LysAsn Ala 165 170 175 Arg Arg Leu Met Asn Leu His Asn Asn Glu Ala Gly ArgLys Val Leu 180 185 190 Glu Asp Arg Met Gln Leu Glu Cys Lys Cys His GlyVal Ser Gly Ser 195 200 205 Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu ProLys Phe Arg Glu Val 210 215 220 Gly His Leu Leu Lys Glu Lys Tyr Asn AlaAla Val Gln Val Glu Val 225 230 235 240 Val Arg Ala Ser Arg Leu Arg GlnPro Thr Phe Leu Arg Ile Lys Gln 245 250 255 Leu Arg Ser Tyr Gln Lys ProMet Glu Thr Asp Leu Val Tyr Ile Glu 260 265 270 Lys Ser Pro Asn Tyr CysGlu Glu Asp Ala Ala Thr Gly Ser Val Gly 275 280 285 Thr Gln Gly Arg LeuCys Asn Arg Thr Ser Pro Gly Ala Asp Gly Cys 290 295 300 Asp Thr Met CysCys Gly Arg Gly Tyr Asn Thr His Gln Tyr Thr Lys 305 310 315 320 Val TrpGln Cys Asn Cys Lys Phe His Trp Cys Cys Phe Val Lys Cys 325 330 335 AsnThr Cys Ser Glu Arg Thr Glu Val Phe Thr Cys Lys 340 345 21 355 PRT Homosapiens human Wingless-type 8A (Wnt-8A) peptide sequence 21 Met Gly AsnLeu Phe Met Leu Trp Ala Ala Leu Gly Ile Cys Cys Ala 1 5 10 15 Ala PheSer Ala Ser Ala Trp Ser Val Asn Asn Phe Leu Ile Thr Gly 20 25 30 Pro LysAla Tyr Leu Thr Tyr Thr Thr Ser Val Ala Leu Gly Ala Gln 35 40 45 Ser GlyIle Glu Glu Cys Lys Phe Gln Phe Ala Trp Glu Arg Trp Asn 50 55 60 Cys ProGlu Asn Ala Leu Gln Leu Ser Thr His Asn Arg Leu Arg Ser 65 70 75 80 AlaThr Arg Glu Thr Ser Phe Ile His Ala Ile Ser Ser Ala Gly Val 85 90 95 MetTyr Ile Ile Thr Lys Asn Cys Ser Met Gly Asp Phe Glu Asn Cys 100 105 110Gly Cys Asp Gly Ser Asn Asn Gly Lys Thr Gly Gly His Gly Trp Ile 115 120125 Trp Gly Gly Cys Ser Asp Asn Val Glu Phe Gly Glu Arg Ile Ser Lys 130135 140 Leu Phe Val Asp Ser Leu Glu Lys Gly Lys Asp Ala Arg Ala Leu Met145 150 155 160 Asn Leu His Asn Asn Arg Ala Gly Arg Leu Ala Val Arg AlaThr Met 165 170 175 Lys Arg Thr Cys Lys Cys His Gly Ile Ser Gly Ser CysSer Ile Gln 180 185 190 Thr Cys Trp Leu Gln Leu Ala Glu Phe Arg Glu MetGly Asp Tyr Leu 195 200 205 Lys Ala Lys Tyr Asp Gln Ala Leu Lys Ile GluMet Asp Lys Arg Gln 210 215 220 Leu Arg Ala Gly Asn Ser Ala Glu Gly HisTrp Val Pro Ala Glu Ala 225 230 235 240 Phe Leu Pro Ser Ala Glu Ala GluLeu Ile Phe Leu Glu Glu Ser Pro 245 250 255 Asp Tyr Cys Thr Cys Asn SerSer Leu Gly Ile Tyr Gly Thr Glu Gly 260 265 270 Arg Glu Cys Leu Gln AsnSer His Asn Thr Ser Arg Trp Glu Arg Arg 275 280 285 Ser Cys Gly Arg LeuCys Thr Glu Cys Gly Leu Gln Val Glu Glu Arg 290 295 300 Lys Thr Glu ValIle Ser Ser Cys Asn Cys Lys Phe Gln Trp Cys Cys 305 310 315 320 Thr ValLys Cys Asp Gln Cys Arg His Val Val Ser Lys Tyr Tyr Cys 325 330 335 AlaArg Ser Pro Gly Ser Ala Gln Ser Leu Gly Arg Val Trp Phe Gly 340 345 350Val Tyr Ile 355 22 351 PRT Homo sapiens human Wingless-type 8B (Wnt-8B)peptide sequence 22 Met Phe Leu Ser Lys Pro Ser Val Tyr Ile Cys Leu PheThr Cys Val 1 5 10 15 Leu Gln Leu Ser His Ser Trp Ser Val Asn Asn PheLeu Met Thr Gly 20 25 30 Pro Lys Ala Tyr Leu Ile Tyr Ser Ser Ser Val AlaAla Gly Ala Gln 35 40 45 Ser Gly Ile Glu Glu Cys Lys Tyr Gln Phe Ala TrpAsp Arg Trp Asn 50 55 60 Cys Pro Glu Arg Ala Leu Gln Leu Ser Ser His GlyGly Leu Arg Ser 65 70 75 80 Ala Asn Arg Glu Thr Ala Phe Val His Ala IleSer Ser Ala Gly Val 85 90 95 Met Tyr Thr Leu Thr Arg Asn Cys Ser Leu GlyAsp Phe Asp Asn Cys 100 105 110 Gly Cys Asp Asp Ser Arg Asn Gly Gln LeuGly Gly Gln Gly Trp Leu 115 120 125 Trp Gly Gly Cys Ser Asp Asn Val GlyPhe Gly Glu Ala Ile Ser Lys 130 135 140 Gln Phe Val Asp Ala Leu Glu ThrGly Gln Asp Ala Arg Ala Ala Met 145 150 155 160 Asn Leu His Asn Asn GluAla Gly Arg Lys Ala Val Lys Gly Thr Met 165 170 175 Lys Arg Thr Cys LysCys His Gly Val Ser Gly Ser Cys Thr Thr Gln 180 185 190 Thr Cys Trp LeuGln Leu Pro Glu Phe Arg Glu Val Gly Ala His Leu 195 200 205 Lys Glu LysTyr His Ala Ala Leu Lys Val Asp Leu Leu Gln Gly Ala 210 215 220 Gly AsnSer Ala Ala Ala Arg Gly Ala Ile Ala Asp Thr Phe Arg Ser 225 230 235 240Ile Ser Thr Arg Glu Leu Val His Leu Glu Asp Ser Pro Asp Tyr Cys 245 250255 Leu Glu Asn Lys Thr Leu Gly Leu Leu Gly Thr Glu Gly Arg Glu Cys 260265 270 Leu Arg Arg Gly Arg Ala Leu Gly Arg Trp Glu Leu Arg Ser Cys Arg275 280 285 Arg Leu Cys Gly Asp Cys Gly Leu Ala Val Glu Glu Arg Arg AlaGlu 290 295 300 Thr Val Ser Ser Cys Asn Cys Lys Phe His Trp Cys Cys AlaVal Arg 305 310 315 320 Cys Glu Gln Cys Arg Arg Arg Val Thr Lys Tyr PheCys Ser Arg Ala 325 330 335 Glu Arg Pro Arg Gly Gly Ala Ala His Lys ProGly Arg Lys Pro 340 345 350 23 417 PRT Homo sapiens human Wingless-type10A (Wnt-10A) peptide sequence 23 Met Gly Ser Ala His Pro Arg Pro TrpLeu Arg Leu Arg Pro Gln Pro 1 5 10 15 Gln Pro Arg Pro Ala Leu Trp ValLeu Leu Phe Phe Leu Leu Leu Leu 20 25 30 Ala Ala Ala Met Pro Arg Ser AlaPro Asn Asp Ile Leu Asp Leu Arg 35 40 45 Leu Pro Pro Glu Pro Val Leu AsnAla Asn Thr Val Cys Leu Thr Leu 50 55 60 Pro Gly Leu Ser Arg Arg Gln MetGlu Val Cys Val Arg His Pro Asp 65 70 75 80 Val Ala Ala Ser Ala Ile GlnGly Ile Gln Ile Ala Ile His Glu Cys 85 90 95 Gln His Gln Phe Arg Asp GlnArg Trp Asn Cys Ser Ser Leu Glu Thr 100 105 110 Arg Asn Lys Ile Pro TyrGlu Ser Pro Ile Phe Ser Arg Gly Phe Arg 115 120 125 Glu Ser Ala Phe AlaTyr Ala Ile Ala Ala Ala Gly Val Val His Ala 130 135 140 Val Ser Asn AlaCys Ala Leu Gly Lys Leu Lys Ala Cys Gly Cys Asp 145 150 155 160 Ala SerArg Arg Gly Asp Glu Glu Ala Phe Arg Arg Lys Leu His Arg 165 170 175 LeuGln Leu Asp Ala Leu Gln Arg Gly Lys Gly Leu Ser His Gly Val 180 185 190Pro Glu His Pro Ala Leu Pro Thr Ala Ser Pro Gly Leu Gln Asp Ser 195 200205 Trp Glu Trp Gly Gly Cys Ser Pro Asp Met Gly Phe Gly Glu Arg Phe 210215 220 Ser Lys Asp Phe Leu Asp Ser Arg Glu Pro His Arg Asp Ile His Ala225 230 235 240 Arg Met Arg Leu His Asn Asn Arg Val Gly Arg Gln Ala ValMet Glu 245 250 255 Asn Met Arg Arg Lys Cys Lys Cys His Gly Thr Ser GlySer Cys Gln 260 265 270 Leu Lys Thr Cys Trp Gln Val Thr Pro Glu Phe ArgThr Val Gly Ala 275 280 285 Leu Leu Arg Ser Arg Phe His Arg Ala Thr LeuIle Arg Pro His Asn 290 295 300 Arg Asn Gly Gly Gln Leu Glu Pro Gly ProAla Gly Ala Pro Ser Pro 305 310 315 320 Ala Pro Gly Ala Pro Gly Pro ArgArg Arg Ala Ser Pro Ala Asp Leu 325 330 335 Val Tyr Phe Glu Lys Ser ProAsp Phe Cys Glu Arg Glu Pro Arg Leu 340 345 350 Asp Ser Ala Gly Thr ValGly Arg Leu Cys Asn Lys Ser Ser Ala Gly 355 360 365 Ser Asp Gly Cys GlySer Met Cys Cys Gly Arg Gly His Asn Ile Leu 370 375 380 Arg Gln Thr ArgSer Glu Arg Cys His Cys Arg Phe His Trp Cys Cys 385 390 395 400 Phe ValVal Cys Glu Glu Cys Arg Ile Thr Glu Trp Val Ser Val Cys 405 410 415 Lys24 389 PRT Homo sapiens human Wingless-type 10B (Wnt-10B) peptidesequence 24 Met Leu Glu Glu Pro Arg Pro Arg Pro Pro Pro Ser Gly Leu AlaGly 1 5 10 15 Leu Leu Phe Leu Ala Leu Cys Ser Arg Ala Leu Ser Asn GluIle Leu 20 25 30 Gly Leu Lys Leu Pro Gly Glu Pro Pro Leu Thr Ala Asn ThrVal Cys 35 40 45 Leu Thr Leu Ser Gly Leu Ser Lys Arg Gln Leu Gly Leu CysLeu Arg 50 55 60 Asn Pro Asp Val Thr Ala Ser Ala Leu Gln Gly Leu His IleAla Val 65 70 75 80 His Glu Cys Gln His Gln Leu Arg Asp Gln Arg Trp AsnCys Ser Ala 85 90 95 Leu Glu Gly Gly Gly Arg Leu Pro His His Ser Ala IleLeu Lys Arg 100 105 110 Gly Phe Arg Glu Ser Ala Phe Ser Phe Ser Met LeuAla Ala Gly Val 115 120 125 Met His Ala Val Ala Thr Ala Cys Ser Leu GlyLys Leu Val Ser Cys 130 135 140 Gly Cys Gly Trp Lys Gly Ser Gly Glu GlnAsp Arg Leu Arg Ala Lys 145 150 155 160 Leu Leu Gln Leu Gln Ala Leu SerArg Gly Lys Ser Phe Pro His Ser 165 170 175 Leu Pro Ser Pro Gly Pro GlySer Ser Pro Ser Pro Gly Pro Gln Asp 180 185 190 Thr Trp Glu Trp Gly GlyCys Asn His Asp Met Asp Phe Gly Glu Lys 195 200 205 Phe Ser Arg Asp PheLeu Asp Ser Arg Glu Ala Pro Arg Asp Ile Gln 210 215 220 Ala Arg Met ArgIle His Asn Asn Arg Val Gly Arg Gln Val Val Thr 225 230 235 240 Glu AsnLeu Lys Arg Lys Cys Lys Cys His Gly Thr Ser Gly Ser Cys 245 250 255 GlnPhe Lys Thr Cys Trp Arg Ala Ala Pro Glu Phe Arg Ala Val Gly 260 265 270Ala Ala Leu Arg Glu Arg Leu Gly Arg Ala Ile Phe Ile Asp Thr His 275 280285 Asn Arg Asn Ser Gly Ala Phe Gln Pro Arg Leu Arg Pro Arg Arg Leu 290295 300 Ser Gly Glu Leu Val Tyr Phe Glu Lys Ser Pro Asp Phe Cys Glu Arg305 310 315 320 Asp Pro Thr Met Gly Ser Pro Gly Thr Arg Gly Arg Ala CysAsn Lys 325 330 335 Thr Ser Arg Leu Leu Asp Gly Cys Gly Ser Leu Cys CysGly Arg Gly 340 345 350 His Asn Val Leu Arg Gln Thr Arg Val Glu Arg CysHis Cys Arg Phe 355 360 365 His Trp Cys Cys Tyr Val Leu Cys Asp Glu CysLys Val Thr Glu Trp 370 375 380 Val Asn Val Cys Lys 385 25 354 PRT Homosapiens human Wingless-type 11 (Wnt-11) peptide sequence 25 Met Arg AlaArg Pro Gln Val Cys Glu Ala Leu Leu Phe Ala Leu Ala 1 5 10 15 Leu GlnThr Gly Val Cys Tyr Gly Ile Lys Trp Leu Ala Leu Ser Lys 20 25 30 Thr ProSer Ala Leu Ala Leu Asn Gln Thr Gln His Cys Lys Gln Leu 35 40 45 Glu GlyLeu Val Ser Ala Gln Val Gln Leu Cys Arg Ser Asn Leu Glu 50 55 60 Leu MetHis Thr Val Val His Ala Ala Arg Glu Val Met Lys Ala Cys 65 70 75 80 ArgArg Ala Phe Ala Asp Met Arg Trp Asn Cys Ser Ser Ile Glu Leu 85 90 95 AlaPro Asn Tyr Leu Leu Asp Leu Glu Arg Gly Thr Arg Glu Ser Ala 100 105 110Phe Val Tyr Ala Leu Ser Ala Ala Thr Ile Ser His Ala Ile Ala Arg 115 120125 Ala Cys Thr Ser Gly Asp Leu Pro Gly Cys Ser Cys Gly Pro Val Pro 130135 140 Gly Glu Pro Pro Gly Pro Gly Asn Arg Trp Gly Arg Cys Ala Asp Asn145 150 155 160 Leu Ser Tyr Gly Leu Leu Met Gly Ala Lys Phe Ser Asp AlaPro Met 165 170 175 Lys Val Lys Lys Thr Gly Ser Gln Ala Asn Lys Leu MetArg Leu His 180 185 190 Asn Ser Glu Val Gly Arg Gln Ala Leu Arg Ala SerLeu Glu Met Lys 195 200 205 Cys Lys Cys His Gly Val Ser Gly Ser Cys SerIle Arg Thr Cys Trp 210 215 220 Lys Gly Leu Gln Glu Leu Gln Asp Val AlaAla Asp Leu Lys Thr Arg 225 230 235 240 Tyr Leu Ser Ala Thr Lys Val ValHis Arg Pro Met Gly Thr Arg Lys 245 250 255 His Leu Val Pro Lys Asp LeuAsp Ile Arg Pro Val Lys Asp Trp Glu 260 265 270 Leu Val Tyr Leu Gln SerSer Pro Asp Phe Cys Met Lys Asn Glu Lys 275 280 285 Val Gly Ser His GlyThr Gln Asp Arg Gln Cys Asn Lys Thr Ser Asn 290 295 300 Gly Ser Asp SerCys Asp Leu Met Cys Cys Gly Arg Gly Tyr Asn Pro 305 310 315 320 Tyr ThrAsp Arg Val Val Glu Arg Cys His Cys Lys Tyr His Trp Cys 325 330 335 CysTyr Val Thr Cys Arg Arg Cys Glu Arg Thr Val Glu Arg Tyr Val 340 345 350Cys Lys 26 389 PRT Homo sapiens human Wingless-type 12 (Wnt-12) peptidesequence 26 Met Leu Glu Glu Pro Arg Pro Arg Pro Pro Pro Ser Gly Leu AlaGly 1 5 10 15 Leu Leu Phe Leu Ala Leu Cys Ser Arg Ala Leu Ser Asn GluIle Leu 20 25 30 Gly Leu Lys Leu Pro Gly Glu Pro Pro Leu Thr Ala Asn ThrVal Cys 35 40 45 Leu Thr Leu Ser Gly Leu Ser Lys Arg Gln Leu Gly Leu CysLeu Arg 50 55 60 Asn Pro Asp Val Thr Ala Ser Ala Leu Gln Gly Leu His IleAla Val 65 70 75 80 His Glu Cys Gln His Gln Leu Arg Asp Gln Arg Trp AsnCys Ser Ala 85 90 95 Leu Glu Gly Gly Gly Arg Leu Pro His His Ser Ala IleLeu Lys Arg 100 105 110 Gly Phe Arg Glu Ser Ala Phe Ser Phe Ser Met LeuAla Ala Gly Val 115 120 125 Met His Ala Val Ala Thr Ala Cys Ser Leu GlyLys Leu Val Ser Cys 130 135 140 Gly Cys Gly Trp Lys Gly Ser Gly Glu GlnAsp Arg Leu Arg Ala Lys 145 150 155 160 Leu Leu Gln Leu Gln Ala Leu SerArg Gly Lys Ser Phe Pro His Ser 165 170 175 Leu Pro Ser Pro Gly Pro GlySer Ser Pro Ser Pro Gly Pro Gln Asp 180 185 190 Thr Trp Glu Trp Gly GlyCys Asn His Asp Met Asp Phe Gly Glu Lys 195 200 205 Phe Ser Arg Asp PheLeu Asp Ser Arg Glu Ala Pro Arg Asp Ile Gln 210 215 220 Ala Arg Met ArgIle His Asn Asn Arg Val Gly Arg Gln Val Val Thr 225 230 235 240 Glu AsnLeu Lys Arg Lys Cys Lys Cys His Gly Thr Ser Gly Ser Cys 245 250 255 GlnPhe Lys Thr Cys Trp Arg Ala Ala Pro Glu Phe Arg Ala Val Gly 260 265 270Ala Ala Leu Arg Glu Arg Leu Gly Arg Ala Ile Phe Ile Asp Thr His 275 280285 Asn Arg Asn Ser Gly Ala Phe Gln Pro Arg Leu Arg Pro Arg Arg Leu 290295 300 Ser Gly Glu Leu Val Tyr Phe Glu Lys Ser Pro Asp Phe Cys Glu Arg305 310 315 320 Asp Pro Thr Met Gly Ser Pro Gly Thr Arg Gly Arg Ala CysAsn Lys 325 330 335 Thr Ser Arg Leu Leu Asp Gly Cys Gly Ser Leu Cys CysGly Arg Gly 340 345 350 His Asn Val Leu Arg Gln Thr Arg Val Glu Arg CysHis Cys Arg Phe 355 360 365 His Trp Cys Cys Tyr Val Leu Cys Asp Glu CysLys Val Thr Glu Trp 370 375 380 Val Asn Val Cys Lys 385 27 391 PRT Homosapiens human Wingless-type 13 (Wnt-13) peptide sequence 27 Met Leu ArgPro Gly Gly Ala Glu Glu Ala Ala Gln Leu Pro Leu Arg 1 5 10 15 Arg AlaSer Ala Pro Val Pro Val Pro Ser Pro Ala Ala Pro Asp Gly 20 25 30 Ser ArgAla Ser Ala Arg Leu Gly Leu Ala Cys Leu Leu Leu Leu Leu 35 40 45 Leu LeuThr Leu Pro Ala Arg Val Asp Thr Ser Trp Trp Tyr Ile Gly 50 55 60 Ala LeuGly Ala Arg Val Ile Cys Asp Asn Ile Pro Gly Leu Val Ser 65 70 75 80 ArgGln Arg Gln Leu Cys Gln Arg Tyr Pro Asp Ile Met Arg Ser Val 85 90 95 GlyGlu Gly Ala Arg Glu Trp Ile Arg Glu Cys Gln His Gln Phe Arg 100 105 110His His Arg Trp Asn Cys Thr Thr Leu Asp Arg Asp His Thr Val Phe 115 120125 Gly Arg Val Met Leu Arg Ser Ser Arg Glu Ala Ala Phe Val Tyr Ala 130135 140 Ile Ser Ser Ala Gly Val Val His Ala Ile Thr Arg Ala Cys Ser Gln145 150 155 160 Gly Glu Leu Ser Val Cys Ser Cys Asp Pro Tyr Thr Arg GlyArg His 165 170 175 His Asp Gln Arg Gly Asp Phe Asp Trp Gly Gly Cys SerAsp Asn Ile 180 185 190 His Tyr Gly Val Arg Phe Ala Lys Ala Phe Val AspAla Lys Glu Lys 195 200 205 Arg Leu Lys Asp Ala Arg Ala Leu Met Asn LeuHis Asn Asn Arg Cys 210 215 220 Gly Arg Thr Ala Val Arg Arg Phe Leu LysLeu Glu Cys Lys Cys His 225 230 235 240 Gly Val Ser Gly Ser Cys Thr LeuArg Thr Cys Trp Arg Ala Leu Ser 245 250 255 Asp Phe Arg Arg Thr Gly AspTyr Leu Arg Arg Arg Tyr Asp Gly Ala 260 265 270 Val Gln Val Met Ala ThrGln Asp Gly Ala Asn Phe Thr Ala Ala Arg 275 280 285 Gln Gly Tyr Arg ArgAla Thr Arg Thr Asp Leu Val Tyr Phe Asp Asn 290 295 300 Ser Pro Asp TyrCys Val Leu Asp Lys Ala Ala Gly Ser Leu Gly Thr 305 310 315 320 Ala GlyArg Val Cys Ser Lys Thr Ser Lys Gly Thr Asp Gly Cys Glu 325 330 335 IleMet Cys Cys Gly Arg Gly Tyr Asp Thr Thr Arg Val Thr Arg Val 340 345 350Thr Gln Cys Glu Cys Lys Phe His Trp Cys Cys Ala Val Arg Cys Lys 355 360365 Glu Cys Arg Asn Thr Val Asp Val His Thr Cys Lys Ala Pro Lys Lys 370375 380 Ala Glu Trp Leu Asp Gln Thr 385 390 28 365 PRT Homo sapienshuman Wingless-type 14 (Wnt-14) peptide sequence 28 Met Leu Asp Gly SerPro Leu Ala Arg Trp Leu Ala Ala Ala Phe Gly 1 5 10 15 Leu Thr Leu LeuLeu Ala Ala Leu Arg Pro Ser Ala Ala Tyr Phe Gly 20 25 30 Leu Thr Gly SerGlu Pro Leu Thr Ile Leu Pro Leu Thr Leu Glu Pro 35 40 45 Glu Ala Ala AlaGln Ala His Tyr Lys Ala Cys Asp Arg Leu Lys Leu 50 55 60 Glu Arg Lys GlnArg Arg Met Cys Arg Arg Asp Pro Gly Val Ala Glu 65 70 75 80 Thr Leu ValGlu Ala Val Ser Met Ser Ala Leu Glu Cys Gln Phe Gln 85 90 95 Phe Arg PheGlu Arg Trp Asn Cys Thr Leu Glu Gly Arg Tyr Arg Ala 100 105 110 Ser LeuLeu Lys Arg Gly Phe Lys Glu Thr Ala Phe Leu Tyr Ala Ile 115 120 125 SerSer Ala Gly Leu Thr His Ala Leu Ala Lys Ala Cys Ser Ala Gly 130 135 140Arg Met Glu Arg Cys Thr Cys Asp Glu Ala Pro Asp Leu Glu Asn Arg 145 150155 160 Glu Ala Trp Gln Trp Gly Gly Cys Gly Asp Asn Leu Lys Tyr Ser Ser165 170 175 Lys Phe Val Lys Glu Phe Leu Gly Arg Arg Ser Ser Lys Asp LeuArg 180 185 190 Ala Arg Val Asp Phe His Asn Asn Leu Val Gly Val Lys ValIle Lys 195 200 205 Ala Gly Val Glu Thr Thr Cys Lys Cys His Gly Val SerGly Ser Cys 210 215 220 Thr Val Arg Thr Cys Trp Arg Gln Leu Ala Pro PheHis Glu Val Gly 225 230 235 240 Lys His Leu Lys His Lys Tyr Glu Thr AlaLeu Lys Val Gly Ser Thr 245 250 255 Thr Asn Glu Ala Ala Gly Glu Ala GlyAla Ile Ser Pro Pro Arg Gly 260 265 270 Arg Ala Ser Gly Ala Gly Gly SerAsp Pro Leu Pro Arg Thr Pro Glu 275 280 285 Leu Val His Leu Asp Asp SerPro Ser Phe Cys Leu Ala Gly Arg Phe 290 295 300 Ser Pro Gly Thr Ala GlyArg Arg Cys His Arg Glu Lys Asn Cys Glu 305 310 315 320 Ser Ile Cys CysGly Arg Gly His Asn Thr Gln Ser Arg Val Val Thr 325 330 335 Arg Pro CysGln Cys Gln Val Arg Trp Cys Cys Tyr Val Glu Cys Arg 340 345 350 Gln CysThr Gln Arg Glu Glu Val Tyr Thr Cys Lys Gly 355 360 365 29 357 PRT Homosapiens human Wingless-type 15 (Wnt-15) peptide sequence 29 Met Arg ProPro Pro Ala Leu Ala Leu Ala Gly Leu Cys Leu Leu Ala 1 5 10 15 Leu ProAla Ala Ala Ala Ser Tyr Phe Gly Leu Thr Gly Arg Glu Val 20 25 30 Leu ThrPro Phe Pro Gly Leu Gly Thr Ala Ala Ala Pro Ala Gln Gly 35 40 45 Gly AlaHis Leu Lys Gln Cys Asp Leu Leu Lys Leu Ser Arg Arg Gln 50 55 60 Lys GlnLeu Cys Arg Arg Glu Pro Gly Leu Ala Glu Thr Leu Arg Asp 65 70 75 80 AlaAla His Leu Gly Leu Leu Glu Cys Gln Phe Gln Phe Arg His Glu 85 90 95 ArgTrp Asn Cys Ser Leu Glu Gly Arg Thr Gly Leu Leu Lys Arg Gly 100 105 110Phe Lys Glu Thr Ala Phe Leu Tyr Ala Val Ser Ser Ala Ala Leu Thr 115 120125 His Thr Leu Ala Arg Ala Cys Ser Ala Gly Arg Met Glu Arg Cys Thr 130135 140 Cys Asp Asp Ser Pro Gly Leu Glu Ser Arg Gln Ala Trp Gln Trp Gly145 150 155 160 Val Cys Gly Asp Asn Leu Lys Tyr Ser Thr Lys Phe Leu SerAsn Phe 165 170 175 Leu Gly Ser Lys Arg Gly Asn Lys Asp Leu Arg Ala ArgAla Asp Ala 180 185 190 His Asn Thr His Val Gly Ile Lys Ala Val Lys SerGly Leu Arg Thr 195 200 205 Thr Cys Lys Cys His Gly Val Ser Gly Ser CysAla Val Arg Thr Cys 210 215 220 Trp Lys Gln Leu Ser Pro Phe Arg Glu ThrGly Gln Val Leu Lys Leu 225 230 235 240 Arg Tyr Asp Ser Ala Val Lys ValSer Ser Ala Thr Asn Glu Ala Leu 245 250 255 Gly Arg Leu Glu Leu Trp AlaPro Ala Arg Gln Gly Ser Leu Thr Lys 260 265 270 Gly Leu Ala Pro Arg SerGly Asp Leu Val Tyr Met Glu Asp Ser Pro 275 280 285 Ser Phe Cys Arg ProSer Lys Tyr Ser Pro Gly Thr Ala Gly Arg Val 290 295 300 Cys Ser Arg GluAla Ser Cys Ser Ser Leu Cys Cys Gly Arg Gly Tyr 305 310 315 320 Asp ThrGln Ser Arg Leu Val Ala Phe Ser Cys His Cys Gln Val Gln 325 330 335 TrpCys Cys Tyr Val Glu Cys Gln Gln Cys Val Gln Glu Glu Leu Val 340 345 350Tyr Thr Cys Lys His 355 30 361 PRT Homo sapiens human Wingless-type 16(Wnt-16) peptide sequence 30 Met Glu Arg His Pro Pro Met Gln Leu Thr ThrCys Leu Arg Glu Thr 1 5 10 15 Leu Phe Thr Gly Ala Ser Gln Lys Thr SerLeu Trp Trp Leu Gly Ile 20 25 30 Ala Ser Phe Gly Val Pro Glu Lys Leu GlyCys Ala Asn Leu Pro Leu 35 40 45 Asn Ser Arg Gln Lys Glu Leu Cys Lys ArgLys Pro Tyr Leu Leu Pro 50 55 60 Ser Ile Arg Glu Gly Ala Arg Leu Gly IleGln Glu Cys Arg Ser Gln 65 70 75 80 Phe Arg His Glu Arg Trp Asn Cys MetIle Thr Ala Ala Ala Thr Thr 85 90 95 Ala Pro Met Gly Ala Ser Pro Leu PheGly Tyr Glu Leu Ser Ser Gly 100 105 110 Thr Lys Glu Thr Ala Phe Ile TyrAla Val Met Ala Ala Gly Leu Val 115 120 125 His Ser Val Thr Arg Ser CysSer Ala Gly Asn Met Thr Glu Cys Ser 130 135 140 Cys Asp Thr Thr Leu GlnAsn Gly Gly Ser Ala Ser Glu Gly Trp His 145 150 155 160 Trp Gly Gly CysSer Asp Asp Val Gln Tyr Gly Met Trp Phe Ser Arg 165 170 175 Lys Phe LeuAsp Phe Pro Ile Gly Asn Thr Thr Gly Lys Glu Asn Lys 180 185 190 Val LeuLeu Ala Met Asn Leu His Asn Asn Glu Ala Gly Arg Gln Ala 195 200 205 ValAla Lys Leu Met Ser Val Asp Cys Arg Cys His Gly Val Ser Gly 210 215 220Ser Cys Ala Val Lys Thr Cys Trp Lys Thr Met Ser Ser Phe Glu Lys 225 230235 240 Ile Gly His Leu Leu Lys Asp Lys Tyr Glu Asn Ser Ile Gln Ile Ser245 250 255 Asp Lys Ile Lys Arg Lys Met Arg Arg Arg Glu Lys Asp Gln ArgLys 260 265 270 Ile Pro Ile His Lys Asp Asp Leu Leu Tyr Val Asn Lys SerPro Asn 275 280 285 Tyr Cys Val Glu Asp Lys Lys Leu Gly Ile Pro Gly ThrGln Gly Arg 290 295 300 Glu Cys Asn Arg Thr Ser Glu Gly Ala Asp Gly CysAsn Leu Leu Cys 305 310 315 320 Cys Gly Arg Gly Tyr Asn Thr His Val ValArg His Val Glu Arg Cys 325 330 335 Glu Cys Lys Phe Ile Trp Cys Cys TyrVal Arg Cys Arg Arg Cys Glu 340 345 350 Ser Met Thr Asp Val His Thr CysLys 355 360 31 318 PRT Homo sapiens human Frizzled-1 peptide sequence 31Met Ala Glu Glu Glu Ala Pro Lys Lys Ser Arg Ala Ala Gly Gly Gly 1 5 1015 Ala Ser Trp Glu Leu Cys Ala Gly Ala Leu Ser Ala Arg Leu Ala Glu 20 2530 Glu Gly Ser Gly Asp Ala Gly Gly Arg Arg Arg Pro Pro Val Asp Pro 35 4045 Arg Arg Leu Ala Arg Gln Leu Leu Leu Leu Leu Trp Leu Leu Glu Ala 50 5560 Pro Leu Leu Leu Gly Val Arg Ala Gln Ala Ala Gly Gln Gly Pro Gly 65 7075 80 Gln Gly Pro Gly Pro Gly Gln Gln Pro Pro Pro Pro Pro Pro Gln Gln 8590 95 Gln Gln Ser Gly Gln Gln Tyr Asn Gly Glu Arg Gly Ile Ser Val Pro100 105 110 Asp His Gly Tyr Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr AspIle 115 120 125 Ala Tyr Asn Gln Thr Ile Met Pro Asn Leu Leu Gly His ThrAsn Gln 130 135 140 Glu Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro LeuVal Lys Val 145 150 155 160 Gln Cys Ser Ala Glu Leu Lys Phe Phe Leu CysSer Met Tyr Ala Pro 165 170 175 Val Cys Thr Val Leu Glu Gln Ala Leu ProPro Cys Arg Ser Leu Cys 180 185 190 Glu Arg Ala Arg Gln Gly Cys Glu AlaLeu Met Asn Lys Phe Gly Phe 195 200 205 Gln Trp Pro Asp Thr Leu Lys CysGlu Lys Phe Pro Val His Gly Ala 210 215 220 Gly Glu Leu Cys Val Gly GlnAsn Thr Ser Asp Lys Gly Thr Pro Thr 225 230 235 240 Pro Ser Leu Leu ProGlu Phe Trp Thr Ser Asn Pro Gln His Gly Gly 245 250 255 Gly Gly His ArgGly Gly Phe Pro Gly Gly Ala Gly Ala Ser Glu Arg 260 265 270 Gly Lys PheSer Cys Pro Arg Ala Leu Lys Val Pro Ser Tyr Leu Asn 275 280 285 Tyr HisPhe Leu Gly Glu Lys Asp Cys Gly Ala Pro Cys Glu Pro Thr 290 295 300 LysVal Tyr Gly Leu Met Tyr Phe Gly Pro Glu Glu Leu Arg 305 310 315 32 242PRT Homo sapiens human Frizzled-2 peptide sequence 32 Met Arg Pro ArgSer Ala Leu Pro Arg Leu Leu Leu Pro Leu Leu Leu 1 5 10 15 Leu Pro AlaAla Gly Pro Ala Gln Phe His Gly Glu Lys Gly Ile Ser 20 25 30 Ile Pro AspHis Gly Phe Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr 35 40 45 Asp Ile AlaTyr Asn Gln Thr Ile Met Pro Asn Leu Leu Gly His Thr 50 55 60 Asn Gln GluAsp Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu Val 65 70 75 80 Lys ValGln Cys Ser Pro Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr 85 90 95 Ala ProVal Cys Thr Val Leu Glu Gln Ala Ile Pro Pro Cys Arg Ser 100 105 110 IleCys Glu Arg Ala Arg Gln Gly Cys Glu Ala Leu Met Asn Lys Phe 115 120 125Gly Phe Gln Trp Pro Glu Arg Leu Arg Cys Glu His Phe Pro Arg His 130 135140 Gly Ala Glu Gln Ile Cys Val Gly Gln Asn His Ser Glu Asp Gly Ala 145150 155 160 Pro Ala Leu Leu Thr Thr Ala Pro Pro Pro Gly Leu Gln Pro GlyAla 165 170 175 Gly Gly Thr Pro Gly Gly Pro Gly Gly Gly Gly Ala Pro ProArg Tyr 180 185 190 Ala Thr Leu Glu His Pro Phe His Cys Pro Arg Val LeuLys Val Pro 195 200 205 Ser Tyr Leu Ser Tyr Lys Phe Leu Gly Glu Arg AspCys Ala Ala Pro 210 215 220 Cys Glu Pro Ala Arg Pro Asp Gly Ser Met PhePhe Ser Gln Glu Glu 225 230 235 240 Thr Arg 33 200 PRT Homo sapienshuman Frizzled-3 peptide sequence 33 Met Ala Met Thr Trp Ile Val Phe SerLeu Trp Pro Leu Thr Val Phe 1 5 10 15 Met Gly His Ile Gly Gly His SerLeu Phe Ser Cys Glu Pro Ile Thr 20 25 30 Leu Arg Met Cys Gln Asp Leu ProTyr Asn Thr Thr Phe Met Pro Asn 35 40 45 Leu Leu Asn His Tyr Asp Gln GlnThr Ala Ala Leu Ala Met Glu Pro 50 55 60 Phe His Pro Met Val Asn Leu AspCys Ser Arg Asp Phe Arg Pro Phe 65 70 75 80 Leu Cys Ala Leu Tyr Ala ProIle Cys Met Glu Tyr Gly Arg Val Thr 85 90 95 Leu Pro Cys Arg Arg Leu CysGln Arg Ala Tyr Ser Glu Cys Ser Lys 100 105 110 Leu Met Glu Met Phe GlyVal Pro Trp Pro Glu Asp Met Glu Cys Ser 115 120 125 Arg Phe Pro Asp CysAsp Glu Pro Tyr Pro Arg Leu Val Asp Leu Asn 130 135 140 Leu Ala Gly GluPro Thr Glu Gly Ala Pro Val Ala Val Gln Arg Asp 145 150 155 160 Tyr GlyPhe Trp Cys Pro Arg Glu Leu Lys Ile Asp Pro Asp Leu Gly 165 170 175 TyrSer Phe Leu His Val Arg Asp Cys Ser Pro Pro Cys Pro Asn Met 180 185 190Tyr Phe Arg Arg Glu Glu Leu Ser 195 200 34 217 PRT Homo sapiens humanFrizzled-4 peptide sequence 34 Met Ala Trp Arg Gly Ala Gly Pro Ser ValPro Gly Ala Pro Gly Gly 1 5 10 15 Val Gly Leu Ser Leu Gly Leu Leu LeuGln Leu Leu Leu Leu Leu Gly 20 25 30 Pro Ala Arg Gly Phe Gly Asp Glu GluGlu Arg Arg Cys Asp Pro Ile 35 40 45 Arg Ile Ser Met Cys Gln Asn Leu GlyTyr Asn Val Thr Lys Met Pro 50 55 60 Asn Leu Val Gly His Glu Leu Gln ThrAsp Ala Glu Leu Gln Leu Thr 65 70 75 80 Thr Phe Thr Pro Leu Ile Gln TyrGly Cys Ser Ser Gln Leu Gln Phe 85 90 95 Phe Leu Cys Ser Val Tyr Val ProMet Cys Thr Glu Lys Ile Asn Ile 100 105 110 Pro Ile Gly Pro Cys Gly GlyMet Cys Leu Ser Val Lys Arg Arg Cys 115 120 125 Glu Pro Val Leu Lys GluPhe Gly Phe Ala Trp Pro Glu Ser Leu Asn 130 135 140 Cys Ser Lys Phe ProPro Gln Asn Asp His Asn His Met Cys Met Glu 145 150 155 160 Gly Pro GlyAsp Glu Glu Val Pro Leu Pro His Lys Thr Pro Ile Gln 165 170 175 Pro GlyGlu Glu Cys His Ser Val Gly Thr Asn Ser Asp Gln Tyr Ile 180 185 190 TrpVal Lys Arg Ser Leu Asn Cys Val Leu Lys Cys Gly Tyr Asp Ala 195 200 205Gly Leu Tyr Ser Arg Ser Ala Lys Glu 210 215 35 233 PRT Homo sapienshuman Frizzled-5 peptide sequence 35 Met Ala Arg Pro Asp Pro Ser Ala ProPro Ser Leu Leu Leu Leu Leu 1 5 10 15 Leu Ala Gln Leu Val Gly Arg AlaAla Ala Ala Ser Lys Ala Pro Val 20 25 30 Cys Gln Glu Ile Thr Val Pro MetCys Arg Gly Ile Gly Tyr Asn Leu 35 40 45 Thr His Met Pro Asn Gln Phe AsnHis Asp Thr Gln Asp Glu Ala Gly 50 55 60 Leu Glu Val His Gln Phe Trp ProLeu Val Glu Ile Gln Cys Ser Pro 65 70 75 80 Asp Leu Arg Phe Phe Leu CysThr Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95 Asp Tyr His Lys Pro Leu ProPro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110 Lys Ala Gly Cys Ser ProLeu Met Arg Gln Tyr Gly Phe Ala Trp Pro 115 120 125 Glu Arg Met Ser CysAsp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135 140 Val Leu Cys MetAsp Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro 145 150 155 160 Arg ProPhe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly Ala Pro 165 170 175 AlaSer Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe Val Cys Lys Cys 180 185 190Arg Glu Pro Phe Val Pro Ile Leu Lys Glu Ser His Pro Leu Tyr Asn 195 200205 Lys Val Arg Thr Gly Gln Val Pro Asn Cys Ala Val Pro Cys Tyr Gln 210215 220 Pro Ser Phe Ser Ala Asp Glu Arg Thr 225 230 36 196 PRT Homosapiens human Frizzled-6 peptide sequence 36 Met Glu Met Phe Thr Phe LeuLeu Thr Cys Ile Phe Leu Pro Leu Leu 1 5 10 15 Arg Gly His Ser Leu PheThr Cys Glu Pro Ile Thr Val Pro Arg Cys 20 25 30 Met Lys Met Ala Tyr AsnMet Thr Phe Phe Pro Asn Leu Met Gly His 35 40 45 Tyr Asp Gln Ser Ile AlaAla Val Glu Met Glu His Phe Leu Pro Leu 50 55 60 Ala Asn Leu Glu Cys SerPro Asn Ile Glu Thr Phe Leu Cys Lys Ala 65 70 75 80 Phe Val Pro Thr CysIle Glu Gln Ile His Val Val Pro Pro Cys Arg 85 90 95 Lys Leu Cys Glu LysVal Tyr Ser Asp Cys Lys Lys Leu Ile Asp Thr 100 105 110 Phe Gly Ile ArgTrp Pro Glu Glu Leu Glu Cys Asp Arg Leu Gln Tyr 115 120 125 Cys Asp GluThr Val Pro Val Thr Phe Asp Pro His Thr Glu Phe Leu 130 135 140 Gly ProGln Lys Lys Thr Glu Gln Val Gln Arg Asp Ile Gly Phe Trp 145 150 155 160Cys Pro Arg His Leu Lys Thr Ser Gly Gly Gln Gly Tyr Lys Phe Leu 165 170175 Gly Ile Asp Gln Cys Ala Pro Pro Cys Pro Asn Met Tyr Phe Lys Ser 180185 190 Asp Glu Leu Glu 195 37 251 PRT Homo sapiens human Frizzled-7peptide sequence 37 Met Arg Asp Pro Gly Ala Ala Val Pro Leu Ser Ser LeuGly Phe Cys 1 5 10 15 Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Ser AlaGly Ala Gly Ala 20 25 30 Gln Pro Tyr His Gly Glu Lys Gly Ile Ser Val ProAsp His Gly Phe 35 40 45 Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp IleAla Tyr Asn Gln 50 55 60 Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn GlnGlu Asp Ala Gly 65 70 75 80 Leu Glu Val His Gln Phe Tyr Pro Leu Val LysVal Gln Cys Ser Pro 85 90 95 Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr AlaPro Val Cys Thr Val 100 105 110 Leu Asp Gln Ala Ile Pro Pro Cys Arg SerLeu Cys Glu Arg Ala Arg 115 120 125 Gln Gly Cys Glu Ala Leu Met Asn LysPhe Gly Phe Gln Trp Pro Glu 130 135 140 Arg Leu Arg Cys Glu Asn Phe ProVal His Gly Ala Gly Glu Ile Cys 145 150 155 160 Val Gly Gln Asn Thr SerAsp Gly Ser Gly Gly Pro Gly Gly Gly Pro 165 170 175 Thr Ala Tyr Pro ThrAla Pro Tyr Leu Pro Asp Leu Pro Phe Thr Ala 180 185 190 Leu Pro Pro GlyAla Ser Asp Gly Lys Gly Arg Pro Ala Phe Pro Phe 195 200 205 Ser Cys ProArg Gln Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe 210 215 220 Leu GlyGlu Arg Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn 225 230 235 240Gly Leu Met Tyr Phe Lys Glu Glu Glu Arg Arg 245 250 38 275 PRT Homosapiens human Frizzled-8 peptide sequence 38 Met Glu Trp Gly Tyr Leu LeuGlu Val Thr Ser Leu Leu Ala Ala Leu 1 5 10 15 Ala Leu Leu Gln Arg SerSer Gly Ala Ala Ala Ala Ser Ala Lys Glu 20 25 30 Leu Ala Cys Gln Glu IleThr Val Pro Leu Cys Lys Gly Ile Gly Tyr 35 40 45 Asn Tyr Thr Tyr Met ProAsn Gln Phe Asn His Asp Thr Gln Asp Glu 50 55 60 Ala Gly Leu Glu Val HisGln Phe Trp Pro Leu Val Glu Ile Gln Cys 65 70 75 80 Ser Pro Asp Leu LysPhe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95 Leu Glu Asp Tyr LysLys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105 110 Arg Ala Lys AlaGly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125 Trp Pro AspArg Met Arg Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140 Asp ThrLeu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala 145 150 155 160Pro Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Gly Glu Gln Pro 165 170175 Pro Ser Gly Ser Gly His Gly Arg Pro Pro Gly Ala Arg Pro Pro His 180185 190 Arg Gly Gly Gly Arg Gly Gly Gly Gly Gly Asp Ala Ala Ala Pro Pro195 200 205 Ala Arg Gly Gly Gly Gly Gly Gly Lys Ala Arg Pro Pro Gly GlyGly 210 215 220 Ala Ala Pro Cys Glu Pro Gly Cys Gln Cys Arg Ala Pro MetVal Ser 225 230 235 240 Val Ser Ser Glu Arg His Pro Leu Tyr Asn Arg ValLys Thr Gly Gln 245 250 255 Ile Ala Asn Cys Ala Leu Pro Cys His Asn ProPhe Phe Ser Gln Asp 260 265 270 Glu Arg Ala 275 39 229 PRT Homo sapienshuman Frizzled-9 peptide sequence 39 Met Ala Val Ala Pro Leu Arg Gly AlaLeu Leu Leu Trp Gln Leu Leu 1 5 10 15 Ala Ala Gly Gly Ala Ala Leu GluIle Gly Arg Phe Asp Pro Glu Arg 20 25 30 Gly Arg Gly Ala Ala Pro Cys GlnAla Val Glu Ile Pro Met Cys Arg 35 40 45 Gly Ile Gly Tyr Asn Leu Thr ArgMet Pro Asn Leu Leu Gly His Thr 50 55 60 Ser Gln Gly Glu Ala Ala Ala GluLeu Ala Glu Phe Ala Pro Leu Val 65 70 75 80 Gln Tyr Gly Cys His Ser HisLeu Arg Phe Phe Leu Cys Ser Leu Tyr 85 90 95 Ala Pro Met Cys Thr Asp GlnVal Ser Thr Pro Ile Pro Ala Cys Arg 100 105 110 Pro Met Cys Glu Gln AlaArg Leu Arg Cys Ala Pro Ile Met Glu Gln 115 120 125 Phe Asn Phe Gly TrpPro Asp Ser Leu Asp Cys Ala Arg Leu Pro Thr 130 135 140 Arg Asn Asp ProHis Ala Leu Cys Met Glu Ala Pro Glu Asn Ala Thr 145 150 155 160 Ala GlyPro Ala Glu Pro His Lys Gly Leu Gly Met Leu Pro Val Ala 165 170 175 ProArg Pro Ala Arg Pro Pro Gly Asp Leu Gly Pro Gly Ala Gly Gly 180 185 190Ser Gly Thr Cys Glu Asn Pro Glu Lys Phe Gln Tyr Val Glu Lys Ser 195 200205 Arg Ser Cys Ala Pro Arg Cys Gly Pro Gly Val Glu Val Phe Trp Ser 210215 220 Arg Arg Asp Lys Asp 225 40 225 PRT Homo sapiens humanFrizzled-10 peptide sequence 40 Met Gln Arg Pro Gly Pro Arg Leu Trp LeuVal Leu Gln Val Met Gly 1 5 10 15 Ser Cys Ala Ala Ile Ser Ser Met AspMet Glu Arg Pro Gly Asp Gly 20 25 30 Lys Cys Gln Pro Ile Glu Ile Pro MetCys Lys Asp Ile Gly Tyr Asn 35 40 45 Met Thr Arg Met Pro Asn Leu Met GlyHis Glu Asn Gln Arg Glu Ala 50 55 60 Ala Ile Gln Leu His Glu Phe Ala ProLeu Val Glu Tyr Gly Cys His 65 70 75 80 Gly His Leu Arg Phe Phe Leu CysSer Leu Tyr Ala Pro Met Cys Thr 85 90 95 Glu Gln Val Ser Thr Pro Ile ProAla Cys Arg Val Met Cys Glu Gln 100 105 110 Ala Arg Leu Lys Cys Ser ProIle Met Glu Gln Phe Asn Phe Lys Trp 115 120 125 Pro Asp Ser Leu Asp CysArg Lys Leu Pro Asn Lys Asn Asp Pro Asn 130 135 140 Tyr Leu Cys Met GluAla Pro Asn Asn Gly Ser Asp Glu Pro Thr Arg 145 150 155 160 Gly Ser GlyLeu Phe Pro Pro Leu Phe Arg Pro Gln Arg Pro His Ser 165 170 175 Ala GlnGlu His Pro Leu Lys Asp Gly Gly Pro Gly Arg Gly Gly Cys 180 185 190 AspAsn Pro Gly Lys Phe His His Val Glu Lys Ser Ala Ser Cys Ala 195 200 205Pro Leu Cys Thr Pro Gly Val Asp Val Tyr Trp Ser Arg Glu Asp Lys 210 215220 Arg 225 41 716 PRT Homo sapiens human Disheveled 3 (Dvl-3) aminoacid sequence 41 Met Gly Glu Thr Lys Ile Ile Tyr His Leu Asp Gly Gln GluThr Pro 1 5 10 15 Tyr Leu Val Lys Leu Pro Leu Pro Ala Glu Arg Val ThrLeu Ala Asp 20 25 30 Phe Lys Gly Val Leu Gln Arg Pro Ser Tyr Lys Phe PhePhe Lys Ser 35 40 45 Met Asp Asp Asp Phe Gly Val Val Lys Glu Glu Ile SerAsp Asp Asn 50 55 60 Ala Lys Leu Pro Cys Phe Asn Gly Arg Val Val Tyr TrpLeu Val Ser 65 70 75 80 Ala Glu Gly Ser His Pro Asp Pro Ala Pro Phe CysAla Asp Asn Pro 85 90 95 Ser Glu Leu Pro Pro Pro Met Glu Arg Thr Gly GlyIle Gly Asp Ser 100 105 110 Arg Pro Pro Ser Phe His Pro His Ala Gly GlyGly Ser Gln Glu Asn 115 120 125 Leu Asp Asn Asp Thr Glu Thr Asp Ser LeuVal Ser Ala Gln Arg Glu 130 135 140 Arg Pro Arg Arg Arg Asp Gly Pro GluHis Ala Thr Arg Leu Asn Gly 145 150 155 160 Thr Ala Lys Gly Glu Arg ArgArg Glu Pro Gly Gly Tyr Asp Ser Ser 165 170 175 Ser Thr Leu Met Ser SerGlu Leu Glu Thr Thr Ser Phe Phe Asp Ser 180 185 190 Asp Glu Asp Asp SerThr Ser Arg Phe Ser Ser Ser Thr Glu Gln Ser 195 200 205 Ser Ala Ser ArgLeu Met Arg Arg His Lys Arg Arg Arg Arg Lys Gln 210 215 220 Lys Val SerArg Ile Glu Arg Ser Ser Ser Phe Ser Ser Ile Thr Asp 225 230 235 240 SerThr Met Ser Leu Asn Ile Ile Thr Val Thr Leu Asn Met Glu Lys 245 250 255Tyr Asn Phe Leu Gly Ile Ser Ile Val Gly Gln Ser Asn Glu Arg Gly 260 265270 Asp Gly Gly Ile Tyr Ile Gly Ser Ile Met Lys Gly Gly Ala Val Ala 275280 285 Ala Asp Gly Arg Ile Glu Pro Gly Asp Met Leu Leu Gln Val Asn Glu290 295 300 Ile Asn Phe Glu Asn Met Ser Asn Asp Asp Ala Val Arg Val LeuArg 305 310 315 320 Glu Ile Val His Lys Pro Gly Pro Ile Thr Leu Thr ValAla Lys Cys 325 330 335 Trp Asp Pro Ser Pro Arg Gly Cys Phe Thr Leu ProArg Ser Glu Pro 340 345 350 Ile Arg Pro Ile Asp Pro Ala Ala Trp Val SerHis Thr Ala Ala Met 355 360 365 Thr Gly Thr Phe Pro Ala Tyr Gly Met SerPro Ser Leu Ser Thr Ile 370 375 380 Thr Ser Thr Ser Ser Ser Ile Thr SerSer Ile Pro Asp Thr Glu Arg 385 390 395 400 Leu Asp Asp Phe His Leu SerIle His Ser Asp Met Ala Ala Ile Val 405 410 415 Lys Ala Met Ala Ser ProGlu Ser Gly Leu Glu Val Arg Asp Arg Met 420 425 430 Trp Leu Lys Ile ThrIle Pro Asn Ala Phe Ile Gly Ser Asp Val Val 435 440 445 Asp Trp Leu TyrHis Asn Val Glu Gly Phe Thr Asp Arg Arg Glu Ala 450 455 460 Arg Lys TyrAla Ser Asn Leu Leu Lys Ala Gly Phe Ile Arg His Thr 465 470 475 480 ValAsn Lys Ile Thr Phe Ser Glu Gln Cys Tyr Tyr Ile Phe Gly Asp 485 490 495Leu Cys Gly Asn Met Ala Asn Leu Ser Leu His Asp His Asp Gly Ser 500 505510 Ser Gly Ala Ser Asp Gln Asp Thr Leu Ala Pro Leu Pro His Pro Gly 515520 525 Ala Ala Pro Trp Pro Met Ala Phe Pro Tyr Gln Tyr Pro Pro Pro Pro530 535 540 His Pro Tyr Asn Pro His Pro Gly Phe Pro Glu Leu Gly Tyr SerTyr 545 550 555 560 Gly Gly Gly Ser Ala Ser Ser Gln His Ser Glu Gly SerArg Ser Ser 565 570 575 Gly Ser Asn Arg Ser Gly Ser Asp Arg Arg Lys GluLys Asp Pro Lys 580 585 590 Ala Gly Asp Ser Lys Ser Gly Gly Ser Gly SerGlu Ser Asp His Thr 595 600 605 Thr Arg Ser Ser Leu Arg Gly Pro Arg GluArg Ala Pro Ser Glu Arg 610 615 620 Ser Gly Pro Ala Ala Ser Glu His SerHis Arg Ser His His Ser Leu 625 630 635 640 Ala Ser Ser Leu Arg Ser HisHis Thr His Pro Ser Tyr Gly Pro Pro 645 650 655 Gly Val Pro Pro Leu TyrGly Pro Pro Met Leu Met Met Pro Pro Pro 660 665 670 Pro Ala Ala Met GlyPro Pro Gly Ala Pro Pro Gly Arg Asp Leu Ala 675 680 685 Ser Val Pro ProGlu Leu Thr Ala Ser Arg Gln Ser Phe Arg Met Ala 690 695 700 Met Gly AsnPro Ser Glu Phe Phe Val Asp Val Met 705 710 715 42 670 PRT Homo sapienshuman Disheveled 1 (Dvl-1) amino acid sequence 42 Met Ala Glu Thr LysIle Ile Tyr His Met Asp Glu Glu Glu Thr Pro 1 5 10 15 Tyr Leu Val LysLeu Pro Val Ala Pro Glu Arg Val Thr Leu Ala Asp 20 25 30 Phe Lys Asn ValLeu Ser Asn Arg Pro Val His Ala Tyr Lys Phe Phe 35 40 45 Phe Lys Ser MetAsp Gln Asp Phe Gly Val Val Lys Glu Glu Ile Phe 50 55 60 Asp Asp Asn AlaLys Leu Pro Cys Phe Asn Gly Arg Val Val Ser Trp 65 70 75 80 Leu Val LeuAla Glu Gly Ala His Ser Asp Ala Gly Ser Gln Gly Thr 85 90 95 Asp Ser HisThr Asp Leu Pro Pro Pro Leu Glu Arg Thr Gly Gly Ile 100 105 110 Gly AspSer Arg Pro Pro Ser Phe His Pro Asn Val Ala Ser Ser Arg 115 120 125 AspGly Met Asp Asn Glu Thr Gly Thr Glu Ser Met Val Ser His Arg 130 135 140Arg Glu Arg Ala Arg Arg Arg Asn Arg Glu Glu Ala Ala Arg Thr Asn 145 150155 160 Gly His Pro Arg Gly Asp Arg Arg Arg Asp Val Gly Leu Pro Pro Asp165 170 175 Ser Ala Ser Thr Ala Leu Ser Ser Glu Leu Glu Ser Ser Ser PheVal 180 185 190 Asp Ser Asp Glu Asp Gly Ser Thr Ser Arg Leu Ser Ser SerThr Glu 195 200 205 Gln Ser Thr Ser Ser Arg Leu Ile Arg Lys His Lys ArgArg Arg Arg 210 215 220 Lys Gln Arg Leu Arg Gln Ala Asp Arg Ala Ser SerPhe Ser Ser Ile 225 230 235 240 Thr Asp Ser Thr Met Ser Leu Asn Ile ValThr Val Thr Leu Asn Met 245 250 255 Glu Arg His His Phe Leu Gly Ile SerIle Val Gly Gln Ser Asn Asp 260 265 270 Arg Gly Asp Gly Gly Ile Tyr IleGly Ser Ile Met Lys Gly Gly Ala 275 280 285 Val Ala Ala Asp Gly Arg IleGlu Pro Gly Asp Met Leu Leu Gln Val 290 295 300 Asn Asp Val Asn Phe GluAsn Met Ser Asn Asp Asp Ala Val Arg Val 305 310 315 320 Leu Arg Glu IleVal Ser Gln Thr Gly Pro Ile Ser Leu Thr Val Ala 325 330 335 Lys Cys TrpAsp Pro Thr Pro Arg Ser Tyr Phe Thr Val Pro Arg Ala 340 345 350 Asp ProVal Arg Pro Ile Asp Pro Ala Ala Trp Leu Ser His Thr Ala 355 360 365 AlaLeu Thr Gly Ala Leu Pro Arg Tyr Glu Leu Glu Glu Ala Pro Leu 370 375 380Thr Val Lys Ser Asp Met Ser Ala Val Val Arg Val Met Gln Leu Pro 385 390395 400 Asp Ser Gly Leu Glu Ile Arg Asp Arg Met Trp Leu Lys Ile Thr Ile405 410 415 Ala Asn Ala Val Ile Gly Ala Asp Val Val Asp Trp Leu Tyr ThrHis 420 425 430 Val Glu Gly Phe Lys Glu Arg Arg Glu Ala Arg Lys Tyr AlaSer Ser 435 440 445 Leu Leu Lys His Gly Phe Leu Arg His Thr Val Asn LysIle Thr Phe 450 455 460 Ser Glu Gln Cys Tyr Tyr Val Phe Gly Asp Leu CysSer Asn Leu Ala 465 470 475 480 Thr Leu Asn Leu Asn Ser Gly Ser Ser GlyThr Ser Asp Gln Asp Thr 485 490 495 Leu Ala Pro Leu Pro His Pro Ala AlaPro Trp Pro Leu Gly Gln Gly 500 505 510 Tyr Pro Tyr Gln Tyr Pro Gly ProPro Pro Cys Phe Pro Pro Ala Tyr 515 520 525 Gln Asp Pro Gly Phe Ser TyrGly Ser Gly Ser Thr Gly Ser Gln Gln 530 535 540 Ser Glu Gly Ser Lys SerSer Gly Ser Thr Arg Ser Ser Arg Arg Ala 545 550 555 560 Pro Gly Arg GluLys Glu Arg Arg Ala Ala Gly Ala Gly Gly Ser Gly 565 570 575 Ser Glu SerAsp His Thr Ala Pro Ser Gly Val Gly Ser Ser Trp Arg 580 585 590 Glu ArgPro Ala Gly Gln Leu Ser Arg Gly Ser Ser Pro Arg Ser Gln 595 600 605 AlaSer Ala Thr Ala Pro Gly Leu Pro Pro Pro His Pro Thr Thr Lys 610 615 620Ala Tyr Thr Val Val Gly Gly Pro Pro Gly Gly Pro Pro Val Arg Glu 625 630635 640 Leu Ala Ala Val Pro Pro Glu Leu Thr Gly Ser Arg Gln Ser Phe Gln645 650 655 Lys Ala Met Gly Asn Pro Cys Glu Phe Phe Val Asp Ile Met 660665 670 43 736 PRT Homo sapiens human Disheveled 2 (Dvl-2) amino acidsequence 43 Met Ala Gly Ser Ser Thr Gly Gly Gly Gly Val Gly Glu Thr LysVal 1 5 10 15 Ile Tyr His Leu Asp Glu Glu Glu Thr Pro Tyr Leu Val LysIle Pro 20 25 30 Val Pro Ala Glu Arg Ile Thr Leu Gly Asp Phe Lys Ser ValLeu Gln 35 40 45 Arg Pro Ala Gly Ala Lys Tyr Phe Phe Lys Ser Met Asp GlnAsp Phe 50 55 60 Gly Val Val Lys Glu Glu Ile Ser Asp Asp Asn Ala Arg LeuPro Cys 65 70 75 80 Phe Asn Gly Arg Val Val Ser Trp Leu Val Ser Ser AspAsn Pro Gln 85 90 95 Pro Glu Met Ala Pro Pro Val His Glu Pro Arg Ala GluLeu Ala Pro 100 105 110 Pro Ala Pro Pro Leu Pro Pro Leu Pro Pro Glu ArgThr Ser Gly Ile 115 120 125 Gly Asp Ser Arg Pro Pro Ser Phe His Pro AsnVal Ser Ser Ser His 130 135 140 Glu Asn Leu Glu Pro Glu Thr Glu Thr GluSer Val Val Ser Leu Arg 145 150 155 160 Arg Glu Arg Pro Arg Arg Arg AspSer Ser Glu His Gly Ala Gly Gly 165 170 175 His Arg Thr Gly Gly Pro SerArg Leu Glu Arg His Leu Ala Gly Tyr 180 185 190 Glu Ser Ser Ser Thr LeuMet Thr Ser Glu Leu Glu Ser Thr Ser Leu 195 200 205 Gly Asp Ser Asp GluGlu Asp Thr Met Ser Arg Phe Ser Ser Ser Thr 210 215 220 Glu Gln Ser SerAla Ser Arg Leu Leu Lys Arg His Arg Arg Arg Arg 225 230 235 240 Lys GlnArg Pro Pro Arg Leu Glu Arg Thr Ser Ser Phe Ser Ser Val 245 250 255 ThrAsp Ser Thr Met Ser Leu Asn Ile Ile Thr Val Thr Leu Asn Met 260 265 270Glu Lys Tyr Asn Phe Leu Gly Ile Ser Ile Val Gly Gln Ser Asn Glu 275 280285 Arg Gly Asp Gly Gly Ile Tyr Ile Gly Ser Ile Met Lys Gly Gly Ala 290295 300 Val Ala Ala Asp Gly Arg Ile Glu Pro Gly Asp Met Leu Leu Gln Val305 310 315 320 Asn Asp Met Asn Phe Glu Asn Met Ser Asn Asp Asp Ala ValArg Val 325 330 335 Leu Arg Asp Ile Val His Lys Pro Gly Pro Ile Val LeuThr Val Ala 340 345 350 Lys Cys Trp Asp Pro Ser Pro Gln Ala Tyr Phe ThrLeu Pro Arg Asn 355 360 365 Glu Pro Ile Gln Pro Ile Asp Pro Ala Ala TrpVal Ser His Ser Ala 370 375 380 Ala Leu Thr Gly Thr Phe Pro Ala Tyr ProGly Ser Ser Ser Met Ser 385 390 395 400 Thr Ile Thr Ser Gly Ser Ser LeuPro Asp Gly Cys Glu Gly Arg Gly 405 410 415 Leu Ser Val His Thr Asp MetAla Ser Val Thr Lys Ala Met Ala Ala 420 425 430 Pro Glu Ser Gly Leu GluVal Arg Asp Arg Met Trp Leu Lys Ile Thr 435 440 445 Ile Pro Asn Ala PheLeu Gly Ser Asp Val Val Asp Trp Leu Tyr His 450 455 460 His Val Glu GlyPhe Pro Glu Arg Arg Glu Ala Arg Lys Tyr Ala Ser 465 470 475 480 Gly LeuLeu Lys Ala Gly Leu Ile Arg His Thr Val Asn Lys Ile Thr 485 490 495 PheSer Glu Gln Cys Tyr Tyr Val Phe Gly Asp Leu Ser Gly Gly Cys 500 505 510Glu Ser Tyr Leu Val Asn Leu Ser Leu Asn Asp Asn Asp Gly Ser Ser 515 520525 Gly Ala Ser Asp Gln Asp Thr Leu Ala Pro Leu Pro Gly Ala Thr Pro 530535 540 Trp Pro Leu Leu Pro Thr Phe Ser Tyr Gln Tyr Pro Ala Pro His Pro545 550 555 560 Tyr Ser Pro Gln Pro Pro Pro Tyr His Glu Leu Ser Ser TyrThr Tyr 565 570 575 Gly Gly Gly Ser Ala Ser Ser Gln His Ser Glu Gly SerArg Ser Ser 580 585 590 Gly Ser Thr Arg Ser Asp Gly Gly Ala Gly Arg ThrGly Arg Pro Glu 595 600 605 Glu Arg Ala Pro Glu Ser Lys Ser Gly Ser GlySer Glu Ser Glu Pro 610 615 620 Ser Ser Arg Gly Gly Ser Leu Arg Arg GlyGly Glu Ala Ser Gly Thr 625 630 635 640 Ser Asp Gly Gly Pro Pro Pro SerArg Gly Ser Thr Gly Gly Ala Pro 645 650 655 Asn Leu Arg Ala His Pro GlyLeu His Pro Tyr Gly Pro Pro Pro Gly 660 665 670 Met Ala Leu Pro Tyr AsnPro Met Met Val Val Met Met Pro Pro Pro 675 680 685 Pro Pro Pro Val ProPro Ala Val Gln Pro Pro Gly Ala Pro Pro Val 690 695 700 Arg Asp Leu GlySer Val Pro Pro Glu Leu Thr Ala Ser Arg Gln Ser 705 710 715 720 Phe HisMet Ala Met Gly Asn Pro Ser Glu Phe Phe Val Asp Val Met 725 730 735 44108 DNA Unknown Organism Description of Unknown Organismhybridoma cellline producing anti-human Wnt1 or Wnt2 monoclonal antibody 44 gac attgtg ctg aca cag tct cct gct tcc tta gct gta tct ctg ggg 48 Asp Ile ValLeu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 cag agggcc acc atc tca tac agg gcc agc aaa agt gtc agt aca tct 96 Gln Arg AlaThr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 ggc tat agttat 108 Gly Tyr Ser Tyr 35 45 36 PRT Unknown Organism Description ofUnknown Organismhybridoma cell line producing anti-human Wnt1 or Wnt2monoclonal antibody 45 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu AlaVal Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser LysSer Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr 35 46 60 DNA UnknownOrganism Description of Unknown Organismhybridoma cell line producinganti-human Wnt1 or Wnt2 monoclonal antibody 46 atg cac tgg aac caa cagaaa cca gga cag cca ccc aga ctc ctc atc 48 Met His Trp Asn Gln Gln LysPro Gly Gln Pro Pro Arg Leu Leu Ile 1 5 10 15 tat ctt gta tcc 60 Tyr LeuVal Ser 20 47 20 PRT Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt1 or Wnt2 monoclonalantibody 47 Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu LeuIle 1 5 10 15 Tyr Leu Val Ser 20 48 21 DNA Unknown Organism Descriptionof Unknown Organismhybridoma cell line producing anti-human Wnt1monoclonal antibody 48 aac cta gaa tct ggg gtc cct 21 Asn Leu Glu SerGly Val Pro 1 5 49 7 PRT Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt1 monoclonalantibody 49 Asn Leu Glu Ser Gly Val Pro 1 5 50 21 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt1 monoclonal antibody 50 gcc agg ttc agt ggc agt ggg 21 Ala Arg PheSer Gly Ser Gly 1 5 51 7 PRT Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt1 monoclonalantibody 51 Ala Arg Phe Ser Gly Ser Gly 1 5 52 120 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt1 monoclonal antibody 52 tct ggg aca gac ttc acc ctc aac atc cat cctgtg gag gag gag gat 48 Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro ValGlu Glu Glu Asp 1 5 10 15 gct gca acc tat tac tgt cag cac att agg gagctt aca cgt tcg gag 96 Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu LeuThr Arg Ser Glu 20 25 30 ggg gga cca agc tga aaaaacggg 120 Gly Gly ProSer 35 53 36 PRT Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt1 monoclonalantibody 53 Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu GluAsp 1 5 10 15 Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr ArgSer Glu 20 25 30 Gly Gly Pro Ser 35 54 108 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt1 monoclonal antibody 54 gac att gtg gtg aca cag tct cct gct tcc ttagct gta tct ctg ggg 48 Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu AlaVal Ser Leu Gly 1 5 10 15 cag agg gcc acc atc tca tac agg gcc agc aaaagt gtc agt aca tct 96 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys SerVal Ser Thr Ser 20 25 30 ggc tat agt tat 108 Gly Tyr Ser Tyr 35 55 36PRT Unknown Organism Description of Unknown Organismhybridoma cell lineproducing anti-human Wnt1 monoclonal antibody 55 Asp Ile Val Val Thr GlnSer Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr IleSer Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr 3556 123 DNA Unknown Organism Description of Unknown Organismhybridomacell line producing anti-human Wnt1 monoclonal antibody 56 tct ggg acagac ttc acc ctc aac atc cat cct gtg gag gag gag gat 48 Ser Gly Thr AspPhe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 1 5 10 15 gct gca acctat tac tgt cag cac att agg gag ctt agc acg ttn cgg 96 Ala Ala Thr TyrTyr Cys Gln His Ile Arg Glu Leu Ser Thr Xaa Arg 20 25 30 agg ggg gag ccaagc tga aataaacgg 123 Arg Gly Glu Pro Ser 35 57 37 PRT Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt1 monoclonal antibody 57 Ser Gly Thr Asp Phe Thr Leu Asn Ile His ProVal Glu Glu Glu Asp 1 5 10 15 Ala Ala Thr Tyr Tyr Cys Gln His Ile ArgGlu Leu Ser Thr Xaa Arg 20 25 30 Arg Gly Glu Pro Ser 35 58 21 DNAUnknown Organism Description of Unknown Organismhybridoma cell lineproducing anti-human Wnt2 monoclonal antibody 58 aac cta gaa tct agg aggtca 21 Asn Leu Glu Ser Arg Arg Ser 1 5 59 7 PRT Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt2 monoclonal antibody 59 Asn Leu Glu Ser Arg Arg Ser 1 5 60 21 DNAUnknown Organism Description of Unknown Organismhybridoma cell lineproducing anti-human Wnt2 monoclonal antibody 60 cct gcc agg ttc agt ggtcag 21 Pro Ala Arg Phe Ser Gly Gln 1 5 61 7 PRT Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt2 monoclonal antibody 61 Pro Ala Arg Phe Ser Gly Gln 1 5 62 134 DNAUnknown Organism Description of Unknown Organismhybridoma cell lineproducing anti-human Wnt2 monoclonal antibody 62 tgg tgt ctg gtg tac agactt cac cct cag aca tcc atg cct gtc gga 48 Trp Cys Leu Val Tyr Arg LeuHis Pro Gln Thr Ser Met Pro Val Gly 1 5 10 15 gga gga gga tgc ctg caacct gat tat ntg tgc agc aca tta ggg agc 96 Gly Gly Gly Cys Leu Gln ProAsp Tyr Xaa Cys Ser Thr Leu Gly Ser 20 25 30 tta cac gtt acg gag ggg ggacca agc tga aaaaacgg 134 Leu His Val Thr Glu Gly Gly Pro Ser 35 40 63 41PRT Unknown Organism Description of Unknown Organismhybridoma cell lineproducing anti-human Wnt2 monoclonal antibody 63 Trp Cys Leu Val Tyr ArgLeu His Pro Gln Thr Ser Met Pro Val Gly 1 5 10 15 Gly Gly Gly Cys LeuGln Pro Asp Tyr Xaa Cys Ser Thr Leu Gly Ser 20 25 30 Leu His Val Thr GluGly Gly Pro Ser 35 40 64 27 DNA Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt1 monoclonalantibody 64 ngttncagcc tgnaggagtc nggtgga 27 65 72 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt1 monoclonal antibody 65 ggattggtgc agcctaaagg gtcattgaaa ctctcatgtgcagcctctgg attcactttt 60 aatacctacg cc 72 66 102 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt1 monoclonal antibody 66 atgaactggg tccgccaggc tccaggaaag ggtttggaatgggttgctcg cataagaact 60 agacgttata attctgcaac atattatgcc gattctgtga aa102 67 100 DNA Unknown Organism Description of Unknown Organismhybridomacell line producing anti-human Wnt1 monoclonal antibody 67 gacaggttcaccatctccag agatgattca cggggcatgc tctatctgca aatgaacaac 60 ttgaaaactgaggacacagc catgtattac tgtgtgaggc 100 68 11 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt2 monoclonal antibody 68 agtcnggacc t 11 69 72 DNA Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt2 monoclonal antibody 69 gag ctg gtg aag cct ggg gct tca gtg aag atgtcc tgc aag gct tct 48 Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met SerCys Lys Ala Ser 1 5 10 15 gga tac aca ttc act gac tat gtt 72 Gly Tyr ThrPhe Thr Asp Tyr Val 20 70 24 PRT Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt2 monoclonalantibody 70 Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys AlaSer 1 5 10 15 Gly Tyr Thr Phe Thr Asp Tyr Val 20 71 75 DNA UnknownOrganism Description of Unknown Organismhybridoma cell line producinganti-human Wnt2 monoclonal antibody 71 tta agc tgg gtg aag cag aga actgga cag ggc ctt gag tgg att gga 48 Leu Ser Trp Val Lys Gln Arg Thr GlyGln Gly Leu Glu Trp Ile Gly 1 5 10 15 gag att tat cct gga tat ggt agtact 75 Glu Ile Tyr Pro Gly Tyr Gly Ser Thr 20 25 72 25 PRT UnknownOrganism Description of Unknown Organismhybridoma cell line producinganti-human Wnt2 monoclonal antibody 72 Leu Ser Trp Val Lys Gln Arg ThrGly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Glu Ile Tyr Pro Gly Tyr GlySer Thr 20 25 73 21 DNA Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt2 monoclonalantibody 73 tac tac aat gag aag ttc aag 21 Tyr Tyr Asn Glu Lys Phe Lys 15 74 7 PRT Unknown Organism Description of Unknown Organismhybridomacell line producing anti-human Wnt2 monoclonal antibody 74 Tyr Tyr AsnGlu Lys Phe Lys 1 5 75 156 DNA Unknown Organism Description of UnknownOrganismhybridoma cell line producing anti-human Wnt2 monoclonalantibody 75 ggc aag gcc aca ctg act gct gac aaa tcc tcc aac aca gcc tacatg 48 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met 15 10 15 cag ctc agc agc ctg aca tct gag gac tct gcg gtc tat ttc tgt gca96 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 20 2530 aga tgg ggg gat tgc ttt tgc tta tct ggg gcc aag gga nct ctg gtc 144Arg Trp Gly Asp Cys Phe Cys Leu Ser Gly Ala Lys Gly Xaa Leu Val 35 40 45anc tgt ctc tgc 156 Xaa Cys Leu Cys 50 76 52 PRT Unknown OrganismDescription of Unknown Organismhybridoma cell line producing anti-humanWnt2 monoclonal antibody 76 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser SerAsn Thr Ala Tyr Met 1 5 10 15 Gln Leu Ser Ser Leu Thr Ser Glu Asp SerAla Val Tyr Phe Cys Ala 20 25 30 Arg Trp Gly Asp Cys Phe Cys Leu Ser GlyAla Lys Gly Xaa Leu Val 35 40 45 Xaa Cys Leu Cys 50 77 12 PRT Homosapiens amino acids 201-212 of human Wnt-1 77 His Asn Asn Glu Ala GlyArg Thr Thr Val Phe Ser 1 5 10 78 14 PRT Homo sapiens amino acids 39-52of human Wnt-1 78 Asn Val Ala Ser Ser Thr Asn Leu Leu Thr Asp Ser LysSer 1 5 10 79 5 PRT Artificial Sequence Description of ArtificialSequencepeptide linker 79 Gly Gly Gly Gly Ser 1 5 80 12 PRT ArtificialSequence Description of Artificial Sequencesynthetic peptidecorresponding to amino acid 201-212 of human Wnt-1 80 Xaa Asn Asn GluAla Gly Arg Thr Thr Val Phe Xaa 1 5 10

What is claimed is:
 1. A method of inhibiting the growth of a cancercell that overexpresses a Wnt protein, the method comprising contactingthe cell with an agent that inhibits binding of the Wnt protein to aFrizzled receptor.
 2. The method of claim 1, wherein the agent is anantibody.
 3. The method of claim 2, wherein the antibody specificallybinds to the Wnt protein.
 4. The method of claim 3, wherein the Wntprotein is Wnt-1.
 5. The method of claim 3, wherein the Wnt protein isWnt-2.
 6. The method of claim 2, wherein the antibody specifically bindsa Frizzled receptor.
 7. The method of claim 6, wherein the Frizzledreceptor is a Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5,Frizzled6, Frizzled7, Frizzled8, Frizzled9, and Frizzled10 receptor. 8.The method of claim 2, wherein the antibody is a monoclonal antibody. 9.The method of claim 8, wherein the antibody is recombinantly produced.10. The method of claim 8, wherein the antibody is a humanized antibody.11. The method of claim 8, wherein the antibody is a single chain Fvfragment (scFv).
 12. The method of claim 1, wherein the cancer cell isin a patient and the step of contacting is carried out by administeringthe agent to the patient.
 13. The method of claim 12, wherein the agentis an antibody.
 14. The method of claim 12, further comprisingadministering to the patient a second therapeutic agent.
 15. The methodof claim 14, wherein the second therapeutic agent is a chemotherapeuticagent.
 16. The method of claim 14, wherein the second therapeutic agentis radiation therapy.
 17. The method of claim 1, wherein the cancer cellis a breast cancer cell, colorectal cancer cell, a lung cancer cell, asarcoma cell, a mesothelioma cell, a cervical cancer cell, an ovarycancer cell, a prostate cancer cell, a pancreatic cancer cell, a gastriccancer cell, an esophageal cancer cell, a head and neck cancer cell, ahepatocellular carcinoma cell, a melanoma cell, a glioma cell, aglioblastoma cell, a leukemia cell, or a lymphoma cell.
 18. An anti-Wntmonoclonal antibody that specifically binds to a peptide of SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:9.
 19. The monoclonal antibody of claim 18,wherein the antibody comprises a V_(H) or V_(L) as shown in FIG.
 7. 20.The monoclonal antibody of claim 18, wherein the V_(H) comprises a CDRof a V_(H) chain shown in FIG.
 7. 21. The monoclonal antibody of claim20, wherein the V_(H) comprises all three of the CDRs of a V_(H) chainshown in FIG.
 7. 22. The monoclonal antibody of claim 18, wherein theV_(L) comprises a CDR of a V_(L) region shown in FIG.
 7. 23. Themonoclonal antibody of claim 22, wherein the V_(L) comprises all threeof the CDRs of a V_(L) region shown in FIG.
 7. 24. A pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and amonoclonal antibody that specifically binds Wnt1 or Wnt2.
 25. Thepharmaceutical composition of claim 24, wherein the antibody is furtherconjugated to an effector component.
 26. The pharmaceutical compositionof claim 24, wherein the effector component is a fluorescent label. 27.The pharmaceutical composition of claim 24, wherein the effectorcomponent is a radioisotope or a cytotoxic chemical.
 28. A method ofscreening for an agent that inhibits the proliferation of a cancer cell,the method comprising contacting the agent with a Dvl protein,determining Dvl protein activity or expression, and identifying acompound that inhibits Dvl protein or activity, thereby identifying anagent that inhibits the proliferation of a cancer cell.
 29. The methodof claim 28, further comprising contacting an identified compound with acancer cell, and selecting the compound that inhibits proliferation ofthe cancer cell.
 30. The method of claim 28, wherein the cancer cell isa lung cancer cell.
 31. A method of inhibiting the growth of a cancercell that overexpresses a Dvl protein, the method comprising contactingthe cell with an agent that inhibits Dvl expression or activity.
 32. Themethod of claim 31, wherein the cancer cell is a lung cancer cell. 33.The method of claim 31, wherein the agent is a small molecule.
 34. Themethod of claim 31, wherein the agent is a siRNA.
 35. A method ofinhibiting the growth of a cancer cell that overexpresses a wnt orFrizzled protein, the method comprising contacting the cell with anagent that binds to the intracellular domain of a Frizzled receptor,thereby inhibiting the binding of the Frizzled receptor to anintracellular protein.